U.S. patent application number 13/137324 was filed with the patent office on 2012-04-19 for trans carotenoids, their synthesis, formulation and uses.
This patent application is currently assigned to Diffusion Pharmaceuticals LLC. Invention is credited to John L. Gainer, Marc Lanz.
Application Number | 20120095099 13/137324 |
Document ID | / |
Family ID | 37053852 |
Filed Date | 2012-04-19 |
United States Patent
Application |
20120095099 |
Kind Code |
A1 |
Gainer; John L. ; et
al. |
April 19, 2012 |
Trans carotenoids, their synthesis, formulation and uses
Abstract
The invention relates to trans carotenoid compounds and salts
thereof as well as compositions thereof, methods for making them,
and uses thereof. These compounds are useful in improving
diffusivity of oxygen between red blood cells and body tissues in
mammals including humans.
Inventors: |
Gainer; John L.;
(Charlottesville, VA) ; Lanz; Marc; (Reitnau,
CH) |
Assignee: |
Diffusion Pharmaceuticals
LLC
Charlottesville
VA
|
Family ID: |
37053852 |
Appl. No.: |
13/137324 |
Filed: |
August 5, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11361054 |
Feb 24, 2006 |
8030350 |
|
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13137324 |
|
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60655422 |
Feb 24, 2005 |
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Current U.S.
Class: |
514/560 ;
560/190; 562/595 |
Current CPC
Class: |
A61P 25/02 20180101;
A61P 43/00 20180101; A61K 9/19 20130101; A61P 35/00 20180101; C07C
69/602 20130101; C07C 57/03 20130101; A61P 9/06 20180101; C07C
57/13 20130101; A61P 9/00 20180101; A61P 25/00 20180101; A61P 29/00
20180101; A61P 3/06 20180101; A61K 31/202 20130101; A61P 9/12
20180101; A61N 5/10 20130101; A61K 45/06 20130101; A61K 31/11
20130101; A61K 41/0038 20130101; A61P 25/28 20180101 |
Class at
Publication: |
514/560 ;
560/190; 562/595 |
International
Class: |
A61K 31/202 20060101
A61K031/202; A61P 9/06 20060101 A61P009/06; C07C 57/13 20060101
C07C057/13; C07C 27/02 20060101 C07C027/02; C07C 69/602 20060101
C07C069/602; A61P 9/12 20060101 A61P009/12 |
Goverment Interests
[0002] This invention was made with Government support under
Contract No. N00014-04-C-0146 awarded by the Office of Naval
Research. The Government has certain rights in the invention.
Claims
1. A compound having the structure: ##STR00028##
2. A compound having the structure: ##STR00029##
3. A compound having the structure: ##STR00030##
4. A compound having the structure: ##STR00031##
5. A compound having the structure: ##STR00032##
6. A compound having the structure: ##STR00033##
7. A compound having the structure: ##STR00034##
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. A lyophilized composition of a bipolar trans carotenoid.
25. A composition as in claim 24 wherein the bipolar trans
carotenoid is TSC.
26. A method of synthesizing a BTCS compound having the formula
YZ-TCRO-ZY where: Y=a cation Z=a polar group which is associated
with the cation, and TCRO=trans carotenoid skeleton, comprising the
steps of: coupling a symmetrical dialdehyde containing conjugated
carbon-carbon double bonds with a Wittig agent, and saponifying the
product of the coupling step.
27. A method as in claim 26 wherein the product of the coupling
step is reacted with a second Wittig agent.
28. A method as in claim 26 wherein the symmetrical dialdehyde
containing conjugated carbon-carbon double bonds is a C10 or C20
dial.
29. A method as in claim 26 wherein the coupling reaction is made
in a pH neutral solvent system.
30. A method as in claim 29 wherein said pH neutral system is a
butylene oxide solvent system.
31. A method as in claim 29 wherein said pH neutral system includes
toluene, or methylene chloride/sodium hydroxide and sodium ethylate
or sodium methylate.
32. A method as in claim 26 wherein the Wittig agent is a
triphenylphosphorane.
33. A method as in claim 32 wherein the Wittig agent is
[3-carbomethoxy-2-buten-1-ylidene]triphenylphosphorane.
34. A method as in claim 26 wherein the Wittig agent is a triphenyl
phosphonium bromide or triphenyl phosphonium chloride or a mixture
of the two.
35. A method as in claim 34 wherein the Wittig agent is D
(2-(Ethoxycarbonyl)-2-buten-4-yl-triphenyl-phosphoniumbromide).
36. A method as in claim 26 wherein the Wittig agent is a C2, C3,
C5, C10 or C15 Wittig ester halogenide.
37. A method as in claim 26 wherein the Wittig agent is a C2, C3,
or C5 phosphonoester.
38. A method as in claim 37 wherein the phosphonoester is triphenyl
phosphono acetate.
39. A method as in claim 26 wherein the coupling of step is made
using butylene oxide as the solvent system.
40. A method as in claim 26 wherein after the coupling step is the
step of isolating the desired product of the coupling reaction.
41. A method as in claim 26 wherein the product is saponified using
a solution of NaOH, LiOH, KOH and methanol, ethanol or isopropanol
as the solvent.
42. A method as in claim 26 wherein the product of the coupling
step is saponified using NaOH and ethanol.
43. A method as in claim 26 wherein the product of the coupling
step is saponified using a solution of NaOH and methanol.
44. A method as in claim 26 wherein the step of saponifying the
product of the coupling step comprises the steps of: a)
solubilizing the coupling product in ethanol, and b) mixing the
solution of step a) with a base.
45. A method as in claim 44 wherein the base is selected from the
group consisting of NaOH, KOH, and LiOH.
46. A method as in claim 44 wherein the coupling product is
saponified using ethanol and NaOH.
47. A method as in claim 26 wherein after the saponifying step, the
desired product is washed with ethanol.
48. A method as in claim 26 wherein after the saponifying step, the
desired product is washed with water.
49. A method as in claim 26 wherein the desired product is TSC.
50. A method of synthesizing a BTC compound having the formula
YZ-TCRO-ZY where: Y=H Z=a polar group which is associated with the
H, and TCRO=trans carotenoid skeleton, comprising the steps of:
coupling a symmetrical dialdehyde containing conjugated
carbon-carbon double bonds with a Wittig agent.
51. A method as in claim 50 wherein the coupling reaction is made
in a pH neutral solvent system.
52. A method as in claim 51 wherein said pH neutral system is a
butylene oxide solvent system.
53. A method as in claim 51 wherein said pH neutral system includes
toluene, or methylene chloride/sodium hydroxide and sodium ethylate
or sodium methylate.
54. A method as in claim 50 wherein the Wittig agent is a
triphenylphosphorane.
55. A method as in claim 54 wherein the Wittig agent is
[3-carbomethoxy-2-buten-1-ylidene]triphenylphosphorane.
56. A method as in claim 50 wherein the Wittig agent is a triphenyl
phosphonium bromide or triphenyl phosphonium chloride or a mixture
thereof.
57. A method as in claim 50 wherein the Wittig agent is a C5 Wittig
ester halogenide.
58. A method as in claim 50 wherein the Wittig agent is D
(2-(Ethoxycarbonyl)-2-buten-4-yl-triphenyl-phosphoniumbromide).
59. A method as in claim 50 wherein the coupling of step is made
using butylene oxide as the solvent system.
60. A method as in claim 50 wherein after the coupling step is the
step of isolating the desired product of the coupling reaction.
61. A method as in claim 60 wherein the desired product is washed
with ethanol.
62. A method as in claim 60 wherein the desired product is washed
with water.
63. A method as in claim 50 wherein the desired product is
crocetin.
64. (canceled)
65. (canceled)
66. (canceled)
67. (canceled)
68. (canceled)
69. (canceled)
70. (canceled)
71. A method of treating hypertension in a mammal comprising
administering to the mammal in need of treatment an amount of a
trans carotenoid sufficient to reduce the hypertension.
72. A method of treating ventricular fibrillations or tachycardia
in a mammal comprising administering to a mammal in need of
treatment an amount of a trans carotenoid sufficient to stop the
ventricular fibrillations or tachycardia.
73. A compound having the structure: ##STR00035##
74. A compound selected from the group consisting of TPC and
TLC.
75. A compound selected from the group consisting of Compounds AA
and CC.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/361,054, filed Feb. 24, 2006, which claims the benefit
of Provisional Application No. 60/655,422, filed Feb. 24, 2005, the
entire contents of which are hereby incorporated by reference in
this application.
[0003] Included in this invention are improved chemical synthesis
methods for making trans carotenoids, bipolar trans carotenoids
(BTC) including bipolar trans carotenoid salts (BTCS) such as trans
sodium crocetinate (TSC), the compounds themselves, methods of
formulating them, administering them and methods of using them.
BACKGROUND OF THE INVENTION
[0004] Carotenoids are a class of hydrocarbons consisting of
isoprenoid units joined in such a manner that their arrangement is
reversed at the center of the molecule. The backbone (skeleton) of
the molecule consists of conjugated carbon-carbon double and single
bonds, and can also have pendant groups. Although it was once
thought that the skeleton of a carotenoid contained 40 carbons, it
has been long recognized that carotenoids can also have carbon
skeletons containing fewer than 40 carbon atoms. The 4 single bonds
that surround a carbon-carbon double bond all lie in the same
plane. If the pendant groups are on the same side of the
carbon-carbon double bond, the groups are designated as cis; if
they are on opposite sides of the carbon-carbon bond, they are
designated as trans. Because of the large number of double bonds,
there are extensive possibilities for geometrical (cis/trans)
isomerism of carotenoids, and isomerization occurs readily in
solution. A series of books which is an excellent reference to many
of the properties, etc. of carotenoids ("Carotenoids", edited by G.
Britton, S. Liaaen-Jensen and H. Pfander, Birkhauser Verlag, Basel,
1995 hereby incorporated by reference in its entirety). Many
carotenoids are nonpolar and, thus, are insoluble in water. These
compounds are extremely hydrophobic which makes their formulation
for biological uses difficult because, in order to solubilize them,
one must use an organic solvent rather than an aqueous solvent.
Other carotenoids are monopolar, and have characteristics of
surfactants (a hydrophobic portion and a hydrophilic polar group).
As such, these compounds are attracted to the surface of an aqueous
solution rather than dissolving in the bulk liquid. A few natural
bipolar carotenoid compounds exist, and these compounds contain a
central hydrophobic portion as well as two polar groups, one on
each end of the molecule. It has been reported ("Carotenoids", Vol.
1A, p. 283) that carotenoid sulphates have "significant solubility
in water of up to 0.4 mg/ml". Other carotenoids that might be
thought of as bipolar are also not very soluble in water. These
include dialdehydes and diketones. A di-pyridine salt of crocetin
has also been reported, but its solubility in water is less than 1
mg/ml at room temperature. Other examples of bipolar carotenoids
are crocetin and crocin (both found in the spice saffron). However,
crocetin is only sparingly soluble in water. In fact, of all of the
natural bipolar carotenoids, only crocin displays significant
solubility in water.
[0005] U.S. Pat. Nos. 4,176,179; 4,070,460; 4,046,880; 4,038,144;
4,009,270; 3,975,519; 3,965,261; 3,853,933; and 3,788,468 (each of
which is hereby incorporated by reference in its entirety) relate
to various uses of crocetin.
[0006] U.S. Pat. No. 6,060,511, relates to trans sodium crocetinate
(TSC) and its uses. The TSC is made by reacting naturally occurring
saffron with sodium hydroxide followed by extractions. The '511
patent covers an extraction method for making a bipolar trans
carotenoid salt (Trans Sodium Crocetinate), a purified composition
obtained from extraction, and various uses of the composition such
as improving oxygen diffusivity and treatment of hemorrhagic
shock.
[0007] PCT Application US03/05521 relates to the chemical synthesis
method for making bipolar trans carotenoid salts, and methods of
using them.
[0008] The information below shows the last few steps of a chemical
synthesis process for TSC described in PCT Application
US03/05521.
##STR00001##
[0009] The complete synthesis procedure for TSC, as described in
the PCT application, arrived at key intermediates, "Compound A" and
"Compound B" via multi-step synthetic processes shown in the two
sets of information below:
##STR00002##
Isomerization of Undesired to Desired Dialdehyde:
##STR00003##
[0011] A common form of therapy for malignant tumors, or cancer, is
irradiation. The radiation administered is in the form of
electromagnetic waves or charged or neutral particles.
Electromagnetic waves are represented by x-rays or gamma rays.
Charged particles take the form of electrons, protons, or heavy
ions, while neutrons are an example of neutral particles. During a
course of therapy, the radiation may be administered by external
beam, an interstitial implant, or a combination of the two. With
irradiation, the rad and Gray are the usual units of measure. A
dose of one rad for any type of radiation results in the absorption
of 100 ergs of energy per gram of target tissue, and one Gray is
equal to 100 rads. Therefore, one centiGray (cGy) is equivalent to
one rad. For the majority of smaller tumors of the head and neck, a
course of radiotherapy consisting of 6000 to 6500cGy over 6 to 6.5
weeks is usually adequate. Doses of 6500 to 7000cGy over 6.5 to 7.5
weeks may be necessary to control larger masses with even higher
doses required for bulky disease. It has been shown that a dose of
5000cGy over 5 weeks will control subclinical disease in 90 to 95%
of patients.
[0012] A viable tumor cell is one in which the capacity for
unlimited division is present. A tumor cell must lose this
reproductive capability to be considered killed. Radiotherapeutic
tumor control is achieved by the elimination of all viable cells
within a tumor, and a given dose of radiation will result in the
death of a certain proportion (not number) of viable cells with
each administration. Therefore, the larger the volume of tumor, the
larger the total dose of radiation required for tumor control. A
tumor cell which has been sterilized or killed with radiotherapy
may not necessarily have been morphologically altered and typically
manifests cell death at the time of mitosis (cell division). It is
important to note that this death may not occur with the first cell
division following irradiation. Several apparently successful cell
cycles may take place before cell death becomes overtly manifest,
but the cell is still considered no longer viable in that its
unlimited reproductive potential has already been lost.
[0013] The radiosensitivity of tumor cells is influenced by many
factors. Not long ago, tumor histology and location were thought to
play major roles in the potential control of tumors with
radiotherapy. There is no doubt that certain tumors are more
difficult to control with radiotherapy, but histology is no longer
felt to be as important. The number of viable tumor cells and the
proportion of hypoxic (lacking oxygen) cells within a tumor are
major contributors to radiosensitivity, and both of these are a
function of the size of a given tumor.
[0014] It has been apparent for many years that oxygen plays an
important role in tumor sensitivity to radiation therapy. That
hypoxic tumor cells are more radioresistant is well-established.
While the mechanism for this phenomenon is incompletely understood,
the presence of oxygen is thought to fix radiation injury within
cells which is labile and would otherwise have been repaired. The
maximum change in radiosensitivity occurs over the range of 0-20 mm
of Hg, a value which is well below the venous oxygen tension.
Significant hypoxia has been demonstrated in experimental solid
tumors, and significant indirect evidence indicates hypoxic
conditions within human tumors as well. Hypoxic conditions may
develop because tumors often outgrow their existing blood
supply.
[0015] Chemotherapy is another method used to treat cancer. Drugs
are administered, such carmustine (BCNU), temozolamide (TMZ),
cisplatin, methotrexate, etc., and these drugs will result in the
eventual death or non-growth of the tumor cells. It has been noted
that chemotherapy, like radiation therapy, is less successful with
hypoxic cells--which frequently occur in tumors.
[0016] High blood pressure, or hypertension, affects about one in
four Americans. This potentially life-threatening condition can
exist virtually without symptoms. Blood pressure is characterized
by two values: the systolic blood pressure and the diastolic blood
pressure. Hypertension is generally defined at a systolic pressure
above 140 mm Hg or a diastolic pressure greater than 90 mm Hg;
however, these definitions change and some physicians feel that
blood pressure should remain at 120/70 all one's life, either
naturally or with the use of antihypertensive medicine.
[0017] In some people, the system that regulates blood pressure
goes awry: arterioles throughout the body stay constricted, driving
up the pressure in the larger blood vessels. Sustained high blood
pressure--above 140/90 mm Hg, according to most experts--is called
hypertension. About 90 percent of all people with high blood
pressure have what is currently called "essential"
hypertension--which is meant to denote that it has no identifiable
cause. In the remaining 10 percent of cases, the elevated blood
pressure is due to kidney disease, diabetes, or another underlying
disorder.
SUMMARY OF THE INVENTION
[0018] The invention relates to a variety of novel trans carotenoid
compounds, as well as many compositions containing a trans
carotenoid compound including compositions comprising a trans
carotenoid and a cyclodextrin.
[0019] The invention also includes a method of synthesizing
carotenoid compounds having the formula:
YZ-TCRO-ZY [0020] where: [0021] Y=H or a cation other than H [0022]
Z=a polar group which is associated with Y, and [0023]
TCRO=symmetric or asymmetric trans carotenoid skeleton, comprising
the steps of: coupling a dialdehyde containing conjugated
carbon-carbon double bonds with a Wittig agent, and optionally
saponifying the product of the coupling step.
[0024] In other embodiments, the invention relates to a method of
treating a tumor in a mammal comprising administering to the mammal
i) a trans carotenoid, and ii) radiation or chemotherapy, as well
as methods for treating hypertension, ventricular fibrillations or
tachycardia, or high lipids in a mammal, comprising administering
to the mammal in need of treatment an effective amount of a trans
carotenoid.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 is Study A Results of Tumor Growth.
[0026] FIG. 2 is Study B Results of Radiation Study.
[0027] FIG. 3 is a graph of TSC, Radiation Separately.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The invention relates to an improved chemical synthesis
method for making trans carotenoids, bipolar trans carotenoids
(BTC), bipolar trans carotenoid salts (BTCS) including trans sodium
crocetinate (TSC), the compounds themselves, methods of formulating
them, and methods of using them. As used herein, the term "bipolar"
means having two polar groups, one at each end of the molecule.
[0029] The new method of synthesis invention, described herewith,
is an improvement of the synthetic process described above, i.e. in
U.S. application Ser. No. 10/647,132, hereby incorporated by
reference in its entirety. In the subject invention, Compound B is
substituted with Compound D. The resulting synthesis proceeds to
the Final Product (e.g. trans sodium crocetinate) via a new
penultimate intermediate, Compound E (shown in the following
information).
##STR00004##
[0030] In the new, improved synthetic method, Compound A
(2,7-dimethylocta-2,4,6-triene-1,8-dial) is combined with Compound
D (2-(Ethoxycarbonyl)-2-buten-4-yl-triphenyl-phosphoniumbromide), a
C5 Wittig ester halogenide. These two react to form diethyl
crocetinate or Compound E (Diethyl
2,6,11,15-Tetramethyl-hexadeca-2E,4E,6E,8E,10E,12E,14E-heptaene-1,16-diot-
ate). Compound E is then saponified (via the use of sodium
hydroxide/ethanol) to form the final, desired product, trans sodium
crocetinate.
Compounds of the Invention
[0031] The subject invention relates to trans carotenoids including
trans carotenoid diesters, dialcohols, diketones and diacids,
bipolar trans carotenoids (BTC), and bipolar trans carotenoid salts
(BTCS) compounds and synthesis of such compounds having the
structure:
YZ-TCRO-ZY
where: [0032] Y (which can be the same or different at the two
ends)=H or a cation other than H, preferably Na.sup.+ or K.sup.+ or
Li.sup.+. Y is advantageously a monovalent metal ion. Y can also be
an organic cation, e.g., R.sub.4N.sup.+, R.sub.3S.sup.+, where R is
H, or C.sub.n H.sub.2n+1 where n is 1-10, advantageously 1-6. For
example, R can be methyl, ethyl, propyl or butyl. [0033] Z (which
can be the same or different at the two ends)=polar group which is
associated with H or the cation. Optionally including the terminal
carbon on the carotenoid (or carotenoid related compound), this
group can be a carboxyl (COO.sup.-) group or a CO group (e.g.
ester, aldehyde or ketone group), or a hydroxyl group. This group
can also be a sulfate group (OSO.sub.3.sup.-) or a monophosphate
group (OPO.sub.3.sup.-), (OP(OH)O.sub.2.sup.-), a diphosphate
group, triphosphate or combinations thereof. This group can also be
an ester group of COOR where the R is C.sub.nH.sub.2n+1. [0034]
TCRO=trans carotenoid or carotenoid related skeleton
(advantageously less than 100 carbons) which is linear, has pendant
groups (defined below), and typically comprises "conjugated" or
alternating carbon-carbon double and single bonds (in one
embodiment, the TCRO is not fully conjugated as in a lycopene). The
pendant groups (X) are typically methyl groups but can be other
groups as discussed below. In an advantageous embodiment, the units
of the skeleton are joined in such a manner that their arrangement
is reversed at the center of the molecule. The 4 single bonds that
surround a carbon-carbon double bond all lie in the same plane. If
the pendant groups are on the same side of the carbon-carbon double
bond, the groups are designated as cis (also known as "Z"); if they
are on the opposite side of the carbon-carbon bond, they are
designated as trans (also known as "E"). Throughout this case, the
isomers will be referred to as cis and trans. [0035] The compounds
of the subject invention are trans. The cis isomer typically is a
detriment--and results in the diffusivity not being increased. In
one embodiment, a cis isomer can be utilized where the skeleton
remains linear. The placement of the pendant groups can be
symmetric relative to the central point of the molecule or can be
asymmetric so that the left side of the molecule does not look the
same as the right side of the molecule either in terms of the type
of pendant group or their spatial relationship with respect to the
center carbon.
[0036] The pendant groups X (which can be the same or different)
are hydrogen (H) atoms, or a linear or branched hydrocarbon group
having 10 or less carbons, advantageously 4 or less, (optionally
containing a halogen), or a halogen. X could also be an ester group
(COO-) or an ethoxy/methoxy group. Examples of X are a methyl group
(CH.sub.3), an ethyl group (C.sub.2H.sub.5), a phenyl or single
aromatic ring structure with or without pendant groups from the
ring, a halogen-containing alkyl group (C1-C10) such as CH.sub.2C1,
or a halogen such as Cl or Br or a methoxy (OCH.sub.3) or ethoxy
(OCH.sub.2 CH.sub.3). The pendant groups can be the same or
different but the pendant groups utilized must maintain the
skeleton as linear.
[0037] Although many carotenoids exist in nature, carotenoid salts
do not. Commonly-owned U.S. Pat. No. 6,060,511 hereby incorporated
by reference in its entirety, relates to trans sodium crocetinate
(TSC). The TSC was made by reacting naturally occurring saffron
with sodium hydroxide followed by extractions that selected
primarily for the trans isomer.
[0038] The presence of the cis and trans isomers of a carotenoid or
carotenoid salt can be determined by looking at the
ultraviolet-visible spectrum for the carotenoid sample dissolved in
an aqueous solution. Given the spectrum, the value of the
absorbence of the highest peak which occurs in the visible wave
length range of 380 to 470 nm (the number depending on the solvent
used and the chain length of the BTC or BTCS. The addition of
pendant groups or differing chain lengths will change this peak
absorbance but someone skilled in the art will recognize the
existence of an absorbance peak in the visible range corresponding
to the conjugated backbone structure of these molecules.) is
divided by the absorbency of the peak which occurs in the UV wave
length range of 220 to 300 nm can be used to determine the purity
level of the trans isomer. When the trans carotenoid diester (TCD)
or BTCS is dissolved in water, the highest visible wave length
range peak will be at between 380 nm to 470 nm (depending on the
exact chemical structure, backbone length and pendant groups) and
the UV wave length range peak will be between 220 to 300 nm
According to M. Craw and C. Lambert, Photochemistry and
Photobiology, Vol. 38 (2), 241-243 (1983) hereby incorporated by
reference in its entirety, the result of the calculation (in that
case crocetin was analyzed) was 3.1, which increased to 6.6 after
purification.
[0039] Performing the Craw and Lambert analysis, using a cuvette
designed for UV and visible wavelength ranges, on the trans sodium
salt of crocetin of commonly owned U.S. Pat. No. 6,060,511 (TSC
made by reacting naturally occurring saffron with sodium hydroxide
followed by extractions which selected primarily for the trans
isomer), the value obtained averages about 6.8. Performing that
test on the synthetic TSC of the subject invention, that ratio is
greater than 7.0 (e.g. 7.0 to 8.5), advantageously greater than 7.5
(e.g. 7.5-8.5), most advantageously greater than 8. The synthesized
material is a "purer" or highly purified trans isomer.
[0040] Asymmetric Compounds
[0041] Some examples of asymmetric compounds include but are not
limited to the following: [0042] 1) From Example 6--Synthesis of
Compound P
[0042] ##STR00005## [0043] 2) From Example 8--Synthesis of Compound
U
[0043] ##STR00006## [0044] 3) From Example 9--Synthesis of Compound
W
##STR00007##
[0045] Those skilled in the art will recognize that asymmetry can
be achieved by spatial placement of pendant groups along the length
of the TCRO chain, or by varying the type of pendant group on each
side of the molecule or both. In addition, as in the case of
symmetric trans carotenoid molecules, asymmetric trans carotenoid
molecules can have varying cations, polar end groups and chain
lengths.
[0046] Intermediate Compounds
[0047] In making carotenoid compounds and their salts, certain
intermediate compounds are synthesized prior to obtaining the final
product.
[0048] For example, in the synthesis for TSC, key intermediates
after the coupling of Compounds A and D in the method of the
subject invention are shown below. First is diethyl crocetinate.
Dimethyl crocetinate can also be substituted for diethyl
crocetinate, as can dipropyl etc. forms of the compound. The
structures of some of these intermediates (for a number of the BTCS
molecules presented in examples herein) are shown following:
##STR00008##
Synthesis of the Compounds of the Invention
[0049] One embodiment of the subject invention relates to
improvements made to the last few steps of the process described in
commonly owned PCT Application US03/05521 and U.S. application Ser.
No. 10/647,132. In the new invention, a C10 dial (shown previously
as Compound A) or a C20 dial for the synthesis of long chain
carotenoids, is reacted with a Wittig salt (C2, C3, C5, C10, C15 or
other) through either a single or double step coupling reaction to
form either a single intermediate or two intermediates (one from
each coupling step). The final intermediate is then saponified to
form the BTCS of desired chain length, symmetry and salt type.
[0050] Specifically, in the synthesis of TSC (described in detail
in Examples 1 and 2 herein), the invention relates to the coupling
reaction involving Compounds A and Compound D (rather than Compound
B) resulting in the production of a penultimate intermediate,
Compound E. Compound E is then converted to TSC via a
saponification reaction with sodium hydroxide. In the invention,
the TSC product that is formed is at a higher yield and purity
(compositional and isomeric) than described in the previous
commonly owned application.
Improvements to the Synthesis of Carotenoids such as Trans Sodium
Crocetinate (TSC) Resulting in Higher Yield and Purity:
[0051] 1) Butylene Oxide Solvent for Reaction System
[0052] In the subject invention, the coupling reaction of Compounds
A and D took place in a butylene oxide solvent system. Butylene
oxide/toluene was used as the reaction vehicle for the coupling.
The yield for this step was routinely between 55-60%. By
comparison, a synthesis described in PCT Application US03/05521
used benzene as the vehicle for the analogous coupling reaction
(between Compounds A and B) with a yield at this step of 33%.
[0053] Butylene oxide was used as the solvent in this coupling
reaction because it is a pH-neutral solvent system for the Wittig
reaction. A pH-neutral system is advantageous because 1) there is
no salt generation in a pH-neutral environment, 2) alcohol species
production is inhibited or eliminated, and 3) precipitation of the
final compound is inhibited. Additionally, the use of butylene
oxide and very high purity starting materials (Compounds A and D)
eliminates the need for a second reaction step using NaOH for the
phosphorus ylide conversion (described in the PCT application). The
coupling step in this invention, was thus reduced to a single
reaction step enhancing the overall product yield.
[0054] Other solvents useful in this step include methylene
chloride/sodium hydroxide and sodium ethylate or sodium
methylate.
[0055] 2) Ethanol in saponification step
[0056] In the second step of the new synthesis method,
saponification of the diester (Compound E) to TSC is conducted
using ethanol as a solvent. The use of an ethanol/sodium hydroxide
vehicle for the reaction mixture resulted in a yield at this step
(uncorrected) of 92.0% and a corrected yield of 80%. The yields
reported here are after isolation. Full conversion of the reactants
was observed.
[0057] The diethyl ester (Compound E) is poorly soluble in water
and in a solvent such as THF used in a synthesis method of PCT
Application US03/05521. Hence, the use of ethanol in the subject
invention, which is a more favorable solvent for the diethyl ester,
reduced reaction times significantly and ultimately led to higher
yields. Another suitable alternative in this step is isopropanol or
methanol.
[0058] The results of the changes made to the synthetic process are
summarized in the table below which shows the purity and yield
differences between the method of the subject invention and a
method of PCT Application US03/05521 and U.S. Ser. No. 10/647,132.
It is important to note that the subject process is via diethyl
crocetinate (Compound E) at the penultimate step while the PCT
Application US03/05521 process is via dimethyl crocetinate
(Compound C) at the same step.
Comparison of the Characteristics of Trans Sodium Crocetinate
Synthesized by Two Methods
TABLE-US-00001 [0059] Previous Method Subject Parameter Material
Material HPLC Purity 94.21% 97.56% (measures compositional purity)
Melting Point 3 endothermic events 27.3-136.7.degree. C. (53.5 J/g)
(by DSC) (rather water loss 141.8-170.9 (1.8 J/g) than melting)
22.0-131.3.degree. C. (35.9 J/g) 134.1-161.5.degree. C. (0.6 J/g)
188.1-227.1.degree. C. (1.1 J/g) UV/Vis Data 421 nm: 1.9425 421 nm:
2.2111 Measure of 254 nm: 0.2387 254 nm: 0.2626 isomeric Ratio
(Absorbance Ratio Absorbance purity (0.001M at 421 nm/ at 421 nm/
sodium Absorbance Absorbance carbonate 254 nm): 8.14 254 nm): 8.42
solution in deionized/ distilled water with final solution pH of
8.0) Karl-Fisher 1.63 wt % 1.89 wt % Water Content Elemental C:
62.4 C: 61.5 Analysis H: 5.9 H: 6.2 Na: 11.8 Na: 13.0 O: 19.4 O:
19.8 Intrinsic Biomodal Single mode Particle Size Distribution
Improved Particle Size Distribution
Ethanol Wash
[0060] In experiments where an ethanol washing step was used, the
final water content of the product was lower, (i.e. 0.5 wt %) vs.
the use of a water washing step where the final product water
content was 1.8 wt %. The use of an ethanol wash reduced the
variability of particle size within the lot, making the
distribution more homogeneous. The distribution for TSC produced
via the method of PCT Application US03/05521 was bimodal whereas
this invention demonstrates a simple normal distribution instead.
This improvement is particularly important because particle size
can influence solubility and the more homogeneous distribution
allows for more uniform solubilities.
[0061] The invention thus, includes a method of synthesizing a
trans carotenoid compound having the formula:
YZ-TCRO-ZY [0062] where: [0063] Y (which can be the same or
different at the two ends)=H or a cation, preferably Na.sup.+ or
K.sup.+ or Li.sup.+. Y is advantageously a monovalent metal ion. Y
can also be an organic cation, e.g., R.sub.4N.sup.+,
R.sub.3S.sup.+, where R is H, or C.sub.n H.sub.2n+1 where n is
1-10, advantageously 1-6. For example, R can be methyl, ethyl,
propyl or butyl. [0064] Z (which can be the same or different at
the two ends)=polar group which is associated with the H or cation.
Optionally including the terminal carbon on the carotenoid (or
carotenoid related compound), this group can be a carboxyl (COO)
group or a CO group (e.g. ester, aldehyde or ketone group), or a
hydroxyl group. This group can also be a sulfate group
(OSO.sub.3.sup.-) or a monophosphate group (OPO.sub.3.sup.-),
(OP(OH)O.sub.2.sup.-), a diphosphate group, triphosphate or
combinations thereof. This group can also be an ester group of COOR
where the R is C.sub.n H.sub.2n+1. [0065] TCRO=trans carotenoid or
carotenoid related skeleton (advantageously less than 100 carbons)
which is linear, has pendant groups (defined below), and typically
comprises "conjugated" or alternating carbon-carbon double and
single bonds (in one embodiment, the TCRO is not fully conjugated
as in a lycopene). The pendant groups are typically methyl groups
but can be other groups as discussed below. In an advantageous
embodiment, the units of the skeleton are joined in such a manner
that their arrangement is reversed at the center of the molecule.
[0066] The 4 single bonds that surround a carbon-carbon double bond
all lie in the same plane. If the pendant groups are on the same
side of the carbon-carbon double bond, the groups are designated as
cis; if they are on the opposite side of the carbon-carbon bond,
they are designated as trans. The compounds of the subject
invention are trans. The cis isomer typically is a detriment--and
results in the diffusivity not being increased. In one embodiment,
a cis isomer can be utilized where the skeleton remains linear. The
placement of the pendant groups can be symmetric relative to the
central point of the molecule or can be asymmetric so that the left
side of the molecule does not look the same as the right side of
the molecule either in terms of the type of pendant group or their
spatial relationship with respect to the center carbon.
[0067] The method comprises coupling a symmetrical dialdehyde
containing conjugated carbon-carbon double bonds with a Wittig
agent such as a triphenylphosphorane e.g.
[3-carbomethoxy-2-buten-1-ylidene]triphenylphosphorane or a
triphenyl phosphonium bromide e.g. a C5 Wittig ester halogenide
such as D
(2-(Ethoxycarbonyl)-2-buten-4-yl-triphenyl-phosphoniumbromide) or a
C2, C3 or C5 phosphonoester such as triphenyl phosphono acetate.
The Wittig agent can also be a triphenyl phosophonium chloride or a
mixture of the bromide and chloride compounds. Either a single or
double step coupling reaction is required depending on the length
of the desired TCRO chain. Larger chain lengths require more than
one coupling reaction with either the same or different Wittig
agent at each step, as demonstrated in the examples herein.
[0068] Advantageously, the coupling reaction is made in a pH
neutral solvent system such as a butylene oxide solvent system
optionally including toluene, or methylene chloride/sodium
hydroxide and sodium ethylate or sodium methylate.
[0069] After the coupling step is the step of isolating the desired
product of the coupling reaction.
[0070] After the coupling step, either a second coupling step is
performed and the product is isolated as described above or the
isolated product from the step above is saponified to form a BTCS
compound. If a second coupling reaction is involved, the product
from this second step is isolated and then saponified. The product
can be saponified using a solution of NaOH, LiOH, KOH and methanol,
ethanol or isopropanol as the solvent.
[0071] After the saponifying step, the desired product can be
washed with ethanol or water. In some cases, methanol or
isopropanol is a suitable washing solvent.
Formulation and Administration of the Pharmaceutical Grade
Compounds and Compositions of the Invention
[0072] In formulating trans carotenoids including BTCSs such as
trans sodium crocetinate (TSC) with other ingredients (excipients),
it is advantageous to: improve the solubility (increase the
concentration of the active agent (e.g. TSC) in solution),
stability, bioavailability and isotonic balance of the BTC, reduce
the pH of an aqueous solution, and/or increase the osmolality of an
aqueous solution. The excipient should act as an additive to
prevent self aggregation of monomeric BTC units in solution, or to
prevent pre-mature precipitation of BTC. The addition of the
excipient should aid in at least one of these aspects. Bipolar
trans carotenoid (BTC) molecules can be formulated in a variety of
ways. A basic formulation is a mixture of the BTC in sterile water,
administered by intravenous injection. This formulation can be
modified through the inclusion of various pharmaceutical
excipients, including the cyclodextrins. These formulations can
also be administered by intravenous injection.
[0073] Any of the above described various liquid formulations can
be freeze-dried (lyophilized) to form a dry powder with enhanced
solubility and stability characteristics. Such powdered forms are
then reconstituted for administration. One method is to
reconstitute the powder in a liquid such as saline or sterile water
for injection and then administer it by intravenous injection. This
method can include the use of a multi-compartment syringe
containing the powder in one compartment and liquid in the other
compartment.
[0074] Similarly, the product can be bottled in a vial containing a
barrier separating the powder from the liquid. Before
administration, the barrier is broken and the components mixed
before intravenous injection.
[0075] In addition to intravenous injection, routes of
administration for specially formulated trans carotenoid molecules
include intramuscular injection, delivery by inhalation, oral
administration and transdermal administration.
[0076] Cyclodextrins
[0077] In order to administer some pharmaceuticals, it is necessary
to add another compound which will aid in increasing the
absorption/solubility/concentration of the active pharmaceutical
ingredient (API). Such compounds are called excipients, and
cyclodextrins are examples of excipients. Cyclodextrins are cyclic
carbohydrate chains derived from starch. They differ from one
another by the number of glucopyranose units in their structure.
The parent cyclodextrins contain six, seven and eight glucopyranose
units, and are referred to as alpha, beta and gamma cyclodextrins
respectively. Cyclodextrins were first discovered in 1891, and have
been used as part of pharmaceutical preparations for several
years.
[0078] Cyclodextrins are cyclic (alpha-1,4)-linked oligosaccharides
of alpha-D-gluco-pyranose containing a relatively hydrophobic
central cavity and hydrophilic outer surface. In the pharmaceutical
industry, cyclodextrins have mainly been used as complexing agents
to increase the aqueous solubility of poorly water-soluble drugs,
and to increase their bioavailability and stability. In addition,
cyclodextrins are used to reduce or prevent gastrointestinal or
ocular irritation, reduce or eliminate unpleasant smells or tastes,
prevent drug-drug or drug-additive interactions, or even to convert
oils and liquid drugs into microcrystalline or amorphous
powders.
[0079] Although the BTC compounds are soluble in water, the use of
the cyclodextrins can increase that solubility even more so that a
smaller volume of drug solution can be administered for a given
dosage.
[0080] There are a number of cyclodextrins that can be used with
the Compounds of the Invention. See for example, U.S. Pat. No.
4,727,064, hereby incorporated by reference in its entirety.
Advantageous cyclodextrins are .gamma.-cyclodextrin,
2-hydroxylpropyl-.gamma.-cyclodextrin and
2-hydroxylpropyl-.beta.-cyclodextrin, or other cyclodextrins which
enhance the solubility of the BTC.
[0081] The use of gamma-cyclodextrin with TSC increases the
solubility of TSC in water by 3-7 times. Although this is not as
large a factor as seen in some other cases for increasing the
solubility of an active agent with a cyclodextrin, it is important
in allowing for the parenteral administration of TSC in smaller
volume dosages to humans (or animals). Dosages of TSC and
gamma-cyclodextrin have resulted in aqueous solutions containing as
much as 44 milligrams of TSC per ml of solution. The solutions need
not be equal-molar. The incorporation of the gamma cyclodextrin
also allows for TSC to be absorbed into the blood stream when
injected intramuscularly. Absorption is quick, and efficacious
blood levels of TSC are reached quickly (as shown in rats).
[0082] The cyclodextrin formulation can be used with other trans
carotenoids and carotenoid salts. The subject invention also
includes novel compositions of carotenoids which are not salts
(e.g. acid forms such as crocetin, crocin or the intermediate
compounds noted above) and a cyclodextrin. In other words, trans
carotenoids which are not salts can be formulated with a
cyclodextrin. Mannitol can be added for osmolality, or the
cyclodextrin BTC mixture can be added to isotonic saline (see
below).
[0083] The amount of the cyclodextran used is that amount which
will contain the trans carotenoid but not so much that it will not
release the trans carotenoid.
Cyclodextrin-Mannitol
[0084] A trans carotenoid such as TSC can be formulated with a
cyclodextrin as noted above and a non-metabolized sugar such as
mannitol (e.g. d-mannitol to adjust the osmotic pressure to be the
same as that of blood). Solutions containing over 20 mg TSC/ml of
solution can be made this way. This solution can be added to
isotonic saline or to other isotonic solutions in order to dilute
it and still maintain the proper osmolality. See Example 12.
[0085] Mannitol/Acetic Acid
[0086] A BTCS such as TSC can be formulated with mannitol such as
d-mannitol, and a mild acid such as acetic acid or citric acid to
adjust the pH. The pH of the solution should be around 8 to 8.5. It
should be close to being an isotonic solution, and, as such, can be
injected directly into the blood stream. See Example 13.
[0087] Water+Saline
[0088] A BTCS such as TSC can be dissolved in water (advantageously
injectable water). This solution can then be diluted with water,
normal saline, Ringer's lactate or phosphate buffer, and the
resulting mixture either infused or injected.
Buffers
[0089] A buffer such as glycine or bicarbonate can be added to the
formulation at a level of about 50 mM for stability of the BCT such
as TSC.
TSC and Gamma-Cyclodextrin
[0090] The ratio of TSC to cyclodextrin is based on
TSC:cyclodextrin solubility data. For example, 20 mg/ml TSC, 8%
gamma cyclodextrin, 50 mM glycine, 2.33% mannitol with pH
8.2+/-0.5, or 10 mg/ml TSC and 4% cyclodextrin, or 5 mg/ml and 2%
cyclodextrin. The ratios of these ingredients can be altered
somewhat, as is obvious to one skilled in this art.
[0091] Mannitol can be used to adjust osmolality and its
concentration varies depending on the concentration of other
ingredients. The glycine is held constant. TSC is more stable at
higher pHs. pH of around 8.2+/-0.5 is required for stability and
physiological compatibility. The use of glycine is compatible with
lyophilization. Alternatively, the TSC and cyclodextrin is
formulated using a 50 mM bicarbonate buffer in place of the
glycine.
Endotoxin Removal of Gamma-Cyclodextrin
[0092] Commercially available pharmaceutical grade cyclodextrin has
endotoxin levels that are incompatible with intravenous injection.
The endotoxin levels must be reduced in order to use the
cyclodextrin in a BTC formulation intended for intravenous
injection.
Lyophilization
[0093] Lyophilization as well as other crystallization methods can
be used to dry the BTC drug.
[0094] The Compounds of the Invention can also be formulated
according to the section on formulations set forth in U.S.
application Ser. No. 10/647,132.
Pulmonary Administration
[0095] TSC has been shown to be absorbed into the blood stream
following pulmonary administration. The incorporation of the
.gamma.-cyclodextrin enhances absorption of TSC into the systemic
circulation--with the overall effect of increasing plasma
clearance. Also, an increase in the injection volume results in
greater TSC absorption and over a longer period of time. Thus, a
larger volume injection of the same dose results in a greater
bioavailability. It has been found that hemorrhagic shock in rats
can be successfully treated by administering TSC via the pulmonary
route.
[0096] Cylodextrins are not required for pulmonary absorption.
Pulmonary studies consisting of TSC in pH'd di-water showed
successful absorption into the blood stream.
Intramuscular Administration
[0097] TSC is not absorbed via an intramuscular route when simply
dissolved in de-ionized water; however, the addition of a
cyclodextrin (as in the formulated drug product) results in
absorption into the blood stream. Administration of
.gamma.-cyclodextrin with TSC resulted in successful absorption
into the systemic circulation. It has been found that hemorrhagic
shock in rats can be successfully treated by administering TSC via
intramuscular injection. Formulation of TSC with propylene glycol,
polyethylene glycol polymers (PEG) and other agents also aids in
absorption into the blood stream when TSC is administrated via
intramuscular injections. These agents can also be used with other
BCTs for intramuscular administration.
Transdermal Administration
[0098] TSC has been shown, in rats, to be absorbed into the blood
stream following transdermal administration when formulated with
cyclodextrins. Formulation of TSC with propylene glycol,
polyethylene glycol polymers (PEG), DMSO and other agents also aid
in absorption into the blood stream when TSC is administrated
transdermally. These agents can also be used with other BCTs for
transdermal administration.
Oral Administration
[0099] TSC has been shown to be absorbed into the blood stream
following oral administration. It was found that the incorporation
of a cyclodextrin such as .gamma.-cyclodextrin with a BCT such as
TSC enhances absorption of TSC into the systemic circulation.
Formulation of a BCT with propylene glycol, polyethylene glycol
polymers (PEG) and other agents also enhances oral absorption into
the blood stream.
Uses of the Compounds and Compositions of the Invention
[0100] The compounds and compositions of the subject invention can
be used to treat a variety of disorders in mammals including
humans. The above Compounds of the Invention including the
intermediate compounds, can be used in the uses below, as well as
in the uses set forth in U.S. application Ser. No. 10/647,132.
Trans Carotenoids and Irradiation of Malignant Tumors
[0101] In order to overcome hypoxia of tumor cells which gives rise
to radioresistance, oxygen therapy is useful. In fact, a quantity
has been defined, which is known as the oxygen enhancement ratio
(OER). Its value indicates that the dose of radiation that results
in a given level of cell survival is greater by a constant factor
under hypoxic conditions than when cells are well-oxygenated. For
most mammalian cells, the OER is 2.5 to 3. In other words, 2.5 to 3
times the dose of radiation required to kill well-oxygenated cells
is necessary to kill hypoxic cells. Thus, increasing the oxygen
transport to tumors allows for lower radiation dosages to "kill"
the malignant cells. This is important in many types of tumors.
[0102] The use of a bipolar trans carotenoid compound such as trans
sodium crocetinate has been shown to increase the amount of oxygen
reaching hypoxic tissues; thus, it is a very useful
radiosensitizer. It allows for reduced radiation dosages to be
used, or it increases the effectiveness of irradiation and allows
for tumor regression and cures. It is useful for any type of cancer
for which radiation is currently used. Radiation therapy is given
to about 60% of cancer patients, and a radiation dosage of about
6000-6500 cGy over several weeks is typically used. A BTC or BTCS
such as TSC can be used in conjunction with the radiation to get a
higher cure rate. In one embodiment, TSC is administered at 0.02 to
2 mg/kg, advantageously 0.05 to 1 mg/kg, before each radiation
dosage.
[0103] Higher dosages would be used for another type of dosing
(e.g. 3 times higher since TSC isn't all absorbed in other
routes).
[0104] In one embodiment, another method such as the use of
hyperbaric oxygen, breathing of pure oxygen gas, or the
administration of another compound such as misonidizole, is done in
addition to administration of a BTC compound such as TSC, to
enhance the effectiveness of the radiation. These additional
methods can also be done with the other uses discussed below (e.g.,
chemo).
[0105] The Compounds of the Invention along with radiation
treatment can be used for treating many types of tumors including:
squamous cell carcinomas, melanomas, lymphomas, sarcomas, sarcoids,
osteosarcomas, tumors associated with skin cancer, breast cancer,
head and neck cancer, gynecological cancer, urological and male
genital cancer, bladder cancer, prostate cancer, bone cancer,
cancers of the endocrine glands, cancers of the alimentary canal
(e.g. colon cancer), cancers of the major digestive glands/organs
(e.g. stomach, liver, pancreas), CNS cancer (including brain
cancers such as a gliomas), and lung cancer.
[0106] Trans Sodium Crocetinate (TSC) has been successfully
employed as a radiation sensitizer for a human carcinoma that was
grafted onto mice. Studies were conducted that concluded that a
dosage of TSC ranging from 0.07 mg/kg to 0.18 mg/kg will enhance
the effect of irradiation on these tumor types.
Trans Carotenoids and Chemotherapy
[0107] Trans carotenoid compounds such as trans sodium crocetinate
have been shown to increase the amount of oxygen reaching hypoxic
tissues; which can make it useful in combination with chemotherapy
of cancer. It allows for an increase in the effectiveness of the
chemotherapy. It is useful for any type of cancer for which
chemotherapy is currently used. Chemotherapy is given to the
majority of cancer patients, with many different types of agents
being used. A BTC or BTCS such as TSC can be used in conjunction
with the chemotherapy to get tumor regression and a higher cure
rate. In one embodiment, TSC is administered at 0.02 to 2 mg/kg,
advantageously 0.05 to 1 mg/kg, before, during or after each
chemotherapeutic agent is dosed intravenously. If dosed via another
route, the dosage will need to be increased by a factor of 2 to 3
to account for the decreased bioavailability.
[0108] The Compounds of the Invention along with chemotherapy can
be used for treating many types of tumors including: squamous cell
carcinomas, melanomas, lymphomas, sarcomas, sarcoids,
osteosarcomas, tumors associated with skin cancer, breast cancer,
head and neck cancer, gynecological cancer, urological and male
genital cancer, bladder cancer, prostate cancer, bone cancer,
cancers of the endocrine glands, cancers of the alimentary canal
(e.g. colon cancer), cancers of the major digestive glands/organs
(e.g. stomach, liver, pancreas), CNS cancer (including brain
cancers such as a gliomas), and lung cancer.
Ventricular Fibrillation
[0109] The heart beats when electrical signals move through it.
Ventricular fibrillation ("V fib") is a condition in which the
heart's electrical activity becomes disordered. When this happens,
the heart's lower (pumping) chambers contract in a rapid,
unsynchronized way. (The ventricles "flutter" rather than beat.)
The heart pumps little or no blood.
[0110] Ventricular fibrillation is a very serious condition.
Collapse and sudden cardiac death will follow in minutes unless
medical help is provided immediately. If treated in time, V fib and
ventricular tachycardia (extremely rapid heartbeat) can be
converted into normal rhythm. The present therapy for this
condition requires shocking the heart with a device called a
defibrillator. Another effective way to correct life-threatening
rhythms is by using an electronic device called an implantable
cardioverter-defibrillator. This device shocks the heart to
normalize the heartbeat if the heart's own electrical signals
become disordered.
[0111] Both ventricular fibrillation and tachycardia can also be
"corrected" using a Compound of the Invention such as trans sodium
crocetinate (TSC). TSC, when injected intravenously during a
preclinical study of myocardial infarction, prevented ventricular
fibrillation. In addition, TSC has been shown to reduce tachycardia
in rats subjected to hemorrhagic shock.
[0112] An advantageous dosage of TSC is 0.02-2 mg/kg and more
advantageously 0.05 to 1 mg/kg if dosed intravenously. If dosed via
another route, the dosage will need to be increased by a factor of
2 to 3 to account for the decreased bioavailability.
Hypertension
[0113] Oxygen consumption in humans declines as they age. In
addition, the incidence of hypertension increases with age. While
not wishing to be limited to a specific theory, it is believed that
these two factors are related, i.e., after tissue oxygen
consumption declines, blood pressure increases so as to provide the
tissue with more oxygen. Thus, if more oxygen were provided by some
other method, blood pressure should decrease. An advantageous
dosage of TSC is 0.02-2 mg/kg and more advantageously 0.05 to 1
mg/kg if dosed intravenously. If dosed via another route, the
dosage will need to be increased by a factor of 2 to 3 to account
for the decreased bioavailability.
[0114] The Compounds of the Invention, such as TSC, lower the
systolic blood pressure as well as lowering of the diastolic
pressure. They can also cause a reduction of the heart rate, and
thus cause a decrease in the pulse rate, which is frequently
elevated in the hypertensive patient.
[0115] An advantageous dosage of TSC for treating hypertension is
0.02-2 mg/kg, and more advantageously 0.05 to 1 mg/kg.
High Lipids
[0116] The Compounds of the Invention, such as TSC, can lower
plasma lipid levels including triglyceride and cholesterol levels.
An advantageous dosage of TSC is 0.02-2 mg/kg and more
advantageously 0.05 to 1 mg/kg if dosed intravenously. If dosed via
another route, the dosage will need to be increased by a factor of
2 to 3 to account for the decreased bioavailability.
Use with Premature Babies
[0117] The Compounds of the Invention, such as TSC, can be used
with premature babies to avoid dulling of mental skills. An
advantageous dosage of TSC is 0.02-2 mg/kg and more advantageously
0.05 to 1 mg/kg if dosed intravenously. If dosed via another route,
the dosage will need to be increased by a factor of 2 to 3 to
account for the decreased bioavailability.
Use During Labor
[0118] The Compounds of the Invention, such as TSC, can be used by
administration to the fetus or mother during labor to avoid oxygen
deprivation of the fetus during labor. Oxygen deprivation of the
fetus during labor can result in brain damage or autism. An
advantageous dosage of TSC is 0.02-2 mg/kg and more advantageously
0.05 to 1 mg/kg if dosed intravenously. If dosed via another route,
the dosage will need to be increased by a factor of 2 to 3 to
account for the decreased bioavailability.
Use after Smoke Inhalation
[0119] The Compounds of the Invention, such as TSC, can be
administered after significant smoke inhalation. An advantageous
dosage of TSC is 0.02-2 mg/kg and more advantageously 0.05 to 1
mg/kg if dosed intravenously. If dosed via another route, the
dosage will need to be increased by a factor of 2 to 3 to account
for the decreased bioavailability.
Fibromyalgia
[0120] The Compounds of the Invention, such as TSC, can be used to
treat fibromyalgia by increasing cellular oxygen levels. An
advantageous dosage of TSC is 0.02-2 mg/kg and more advantageously
0.05 to 1 mg/kg if dosed intravenously. If dosed via another route,
the dosage will need to be increased by a factor of 2 to 3 to
account for the decreased bioavailability.
[0121] The following Examples are illustrative, but not limiting of
the compounds, compositions and methods of the present invention.
Other suitable modifications and adaptations of a variety of
conditions and parameters normally encountered which are obvious to
those skilled in the art are within the spirit and scope of this
invention.
EXAMPLES
List of Abbreviations For Examples
[0122] a/a [%] Relative purity in % AP Aqueous layer Approx.
Approximately
COA Certificate of Analysis
[0123] corr. Corrected
d Days
DCM Dichloromethane
DSC Differential Scanning Calorimetry
[0124] E-No. Reference number for each individual compound
Eq Equivalents
[0125] EtOAc Ethyl acetate FW Formula weight GMP Good manufacturing
principles
h Hour
[0126] H-NMR Hydrogen nuclear magnetic resonance HPLC High pressure
liquid chromatography HV Herstellungsvorschrift (Synthesis
procedure)
IPC In-Process-Control
[0127] IT Inner temperature JT Jacket temperature LC-MS Liquid
chromatography-mass spectrometry
MeOH Methanol
min Minutes
[0128] ML Mother liquor MOR Master operation record nc Not
corrected OP Organic layer RT Room temperature (ca. 22.degree. C.)
sat. Saturated soln. Solution sm Starting material Temp.
Temperature TFA. Trifluoroacetic acid Th. Theoretical TPPO
Triphenylphosphine oxide TLC Thin layer chromatography TSC Trans
sodium crocetinate (C-013229) UV Ultraviolet spectroscopy y.
Yield
Example 1
Small-Scale Synthesis of 165 g TSC
Overview of Chemical Reaction
##STR00009##
[0129] Synthesis of Compound E (Diethyl
2,6,11,15-Tetramethyl-hexadeca-2E,4E,6E,8E,10E,12E,14E-heptaene-1,16-diot-
ate)
TABLE-US-00002 ##STR00010## [0130] ##STR00011## Size: 130 g
Compound A 0.792 mol (nc) Th. Quantity: 304.4 g Compound E Pr.
Quantity: 139.6 g Compound D + 0.736 mol (nc) 143.2 Compound E
Yield (nc): 92.9% Purity: 93.68% + 96.99% a/a HPLC Molecular Pos.
Reagent Wt. Eq. Quantity Unit 1 Compound A 164.20 1.0 130 g 2
Compound D 444.01.sup.1 3.0 1052 g 3 Butylene oxide 0.64 L 4
Toluene 1.27 L 5 Ethanol 0.15 L 6 Methylcyclohexane 0.79 L 7
Butylene oxide 0.20 L 8 Toluene 0.50 L 9 Ethanol 0.10 L 10
Methylcyclohexane 0.40 L 11 Methanol 1.00 L .sup.1The used material
was a salt mixture containing 48% bromide and 52% chloride
according to the CoA of the supplier. Thus, the real mass was
different from the theoretical one as depicted on the formula
scheme.
Procedure
[0131] 1. A flask was evacuated and purged with nitrogen. [0132] 2.
The flask was charged with Compound A (1) and Compound D (2) at
JT=20.degree. C. [0133] 3. Butylene oxide (3) and toluene (4) were
charged to the flask at JT=20.degree. C. The flask was evacuated
and purged with nitrogen twice. The reaction mixture was warmed to
JT=100.degree. C. A homogenous solution was obtained. [0134] 4. The
solution was stirred at JT=100.degree. C. for 6.5 h. (IT was about
93.degree. C.). [0135] 5. A sample for an IPC was taken.
TABLE-US-00003 [0135] IPC1#2 No aldehyde signals were detected
(.sup.1H-NMR), see Note 1.
[0136] 6. The mixture was slowly cooled to IT=20.degree. C. (15 h).
[0137] 7. A red suspension was formed. The suspension was cooled to
IT=1.degree. C. within 2 h. [0138] 8. The suspension was filtered
on a filter dryer within for a few minutes. [0139] 9. Cold ethanol
(5), at 2.degree. C., was used to rinse the flask. The rinse
solution was transferred to the filter dryer. [0140] 10. The filter
cake was washed with methylcyclohexane (6) at 22.degree. C. [0141]
11. The filter cake was dried on a rotary evaporator for 5 h. at
55.degree. C. [0142] 12. 139.6 g Compound E was obtained as a red
solid. The identity was confirmed by .sup.1H-NMR. The purity was
93.68% a/a as determined by HPLC. In addition, 2.90+3.00% cis
isomers were also observed. The yield (nc) was 45.9%. [0143] 13.
The mother liquor (about 3 L) was concentrated to 40% of its volume
(still a red solution) and stirred at JT=100.degree. C. for 15 h.
(IT was about 100.degree. C.). [0144] 14. A red suspension was
formed. A sample for an IPC was taken.
TABLE-US-00004 [0144] IPC2#1 Little or no cis isomer was detectable
(.sup.1H-NMR).
[0145] 15. The mixture was diluted with butylene oxide (7) and
toluene (8, still a suspension) and cooled to IT=2.9.degree. C.
within 3.5 h. [0146] 16. The suspension was filtered on a filter
dryer within 10 min. [0147] 17. Cold ethanol (9), about 2.degree.
C., was used to rinse the flask. The rinse solution was transferred
to the filter dryer. [0148] 18. The filter cake was washed with
methylcyclohexane (10) at 18.degree. C. [0149] 19. The filter cake
was dried on a rotary evaporator for 15 h. at 50.degree. C. [0150]
20. 384.5 g crude product was obtained as a red solid. An
.sup.1H-NMR revealed the presence of TPPO in addition to the
desired product. [0151] 21. The crude product was treated with
methanol (11) and stirred for 30 min. at JT=60.degree. C. [0152]
22. The suspension was cooled to JT=0.degree. C. within 60 min.
[0153] 23. The suspension was filtered on a filter dryer within a
few minutes. [0154] 24. Methanol (11) was used to rinse the flask.
The rinse solution was transferred to the filter dryer. [0155] 25.
The filter cake was dried on a rotary evaporator for 2 h. at
55.degree. C. [0156] 26. 143.2 g crude product was obtained as a
red solid. The identity was confirmed by .sup.1H-NMR. The purity
was 96.99% a/a by HPLC. In addition, 1.02+1.26% cis isomers were
observed. The yield (nc) was 47.0%. The .sup.1H-NMR showed about
11.5% TPPO.
Notes
[0156] [0157] 1) A first IPC was taken after 2.5 h. This showed a
complete consumption of the aldehyde and additional cis isomer. The
ratio of isomers improved with the time for the reaction.
Sample Preparation
TABLE-US-00005 [0158] In-Process-Control Conversion: About 0.5 ml
of the reaction (IPC): mixture were taken, evaporated and analysed
by .sup.1H-NMR or HPLC. Purity: 6-7 mg of the product were analysed
by HPLC; method HPLC-TSC-M1.1. .sup.1H-NMR's 5-10 mg of the product
were dissolved in 0.9 ml CDCl.sub.3 (internal standard: TMS) for
NMR spectroscopy.
Synthesis of TSC from Compound E
TABLE-US-00006 ##STR00012## ##STR00013## Size: 185 g Compound A
0.481 mol (nc) Th. Quantity: 179.2 g TSC Pr. Quantity: 164.9 g TSC
0.443 mol (nc) Yield (nc): 92.0% Purity: 97.56% a/a HPLC Molecular
Pos. Reagent Wt. Eq. Quantity Unit 1 Compound E 384.51 1.0 139 + g
46.sup.2 g 2 Ethanol 0.72 L 3 30% NaOH 0.72 L 4 Water 2.21 L 5
Water 1.92 L 6 Water 1.92 L 7 Water 1.92 .sup.2The exact quantity
of starting material was slightly lower than depicted, because the
second portion (46 g) contained about 11.5% TPPO in it according to
the .sup.1H-NMR.
Procedure
[0159] 1. A flask was evacuated and purged with nitrogen. [0160] 2.
The flask was charged with Compound E (1) at JT=20.degree. C.
[0161] 3. Ethanol (2) and 30% NaOH (3) were charged to the flask at
JT=20.degree. C. The flask was evacuated and purged with nitrogen
twice. The reaction mixture was warmed to JT=90.degree. C. A thick,
orange suspension was obtained. [0162] 4. The suspension was
stirred at JT=90.degree. C. for 47 h. (IT about 77.degree. C.).
[0163] 5. The mixture was cooled to IT=21.degree. C. within 16 h. A
sample for an IPC was taken.
TABLE-US-00007 [0163] IPC1#1 98.2% conversion monoester to TSC
(HPLC); no diester (Compound E) was detected.
[0164] 6. The mixture was diluted with water (4). [0165] 7. The
suspension was filtered on a filter dryer within 50 min. [0166] 8.
Cold water (5), at 3.degree. C., was used to rinse the flask and to
wash the filter cake. [0167] 9. The filter cake was washed further
with cold water (6+7), at 5.degree. C. and 2.degree. C. [0168] 10.
The filter cake was dried on a rotary evaporator for 20 h. at
55.degree. C. [0169] 11. 164.9 g TSC was obtained as an orange
solid. The identity was confirmed by .sup.1H-NMR. The purity was
97.56% a/a as measured by HPLC. The yield (nc) was 92.0%.
[0170] The water content was determined to be 1.89% w/w, the UV
ratio 421 nm to 254 nm was 8.42. Anal Calculated for
C.sub.20H.sub.22O.sub.4Na.sub.2--0.5H.sub.2O--0.2NaOH: C, 61.41; H,
6.03; Na, 12.93; 0, 19.63. Found: C, 61.5; H, 6.2; Na, 13.0; 0,
19.8.
Sample Preparation
TABLE-US-00008 [0171] In-Process-Control Conversion: About 0.5 ml
of the reaction (IPC): mixture were taken and analysed by HPLC.
Purity: 6-7 mg of the product were analysed by HPLC; method
HPLC-TSC-M1.1. .sup.1H-NMR's 5-10 mg of the product were dissolved
in 0.9 ml D.sub.2O for NMR spectroscopy.
Example 2
Production of Larger-Scale (2 Kg) TSC Under cGMP Conditions
##STR00014##
[0172] Raw Materials
[0173] The quality of the starting materials (Compounds A and D)
were verified and testing results are shown below:
TABLE-US-00009 HPLC-Purity Lot-No. (% a/a) Appearance Identity by
IR E-027211-002 95.22 Yellow Powder Complies (UQ40112015; C10
dial,; Compound A) E-027684-001 94.70 Yellow Powder Complies
(UQ40112015; C10 dial,; Compound A) E-027684-002 94.60 Yellow
Powder Complies (UQ40112015; C10 dial,; Compound A) E-027758-001-
97.88 White Powder Complies E-027758-003, E-027685-001-
E-027685-021 (UE00401004; C5 Wittig ester,; Compound D)
[0174] All materials fulfilled the given specifications (Compound
D: .gtoreq.97.0%; Compound A: .gtoreq.94.0%). Furthermore, all
materials were prepared synthetically without the use of any animal
components or any components derived from animal products.
[0175] The C-10 dial (Compound A) was a yellow crystalline powder.
The phosphorane, Compound D, was a white to yellow powder. The
identities were checked by .sup.1H-NMR.
Synthesis of Compound E
TABLE-US-00010 ##STR00015## [0176] ##STR00016## Size: 2.99 kg
Compound A 18.21 mol (nc) Th. Quantity: 7.00 kg Compound E Pr.
Quantity: 3.79 kg Compound E + 11.69 mol (nc) 705 g Compound E
Yield (nc): 64.2% Purity: 93.70% + 90.01% a/a HPLC Molecular Pos.
Reagent Wt. Eq. Quantity Unit 1 Compound A 164.20 1.0 2.99 kg 2
Compound D .sup. 444.01.sup.3 3.0 24.04 kg 3 Butylene oxide 15 L 4
Toluene 30 L 5 Butylene oxide 9 L 6 Ethanol 4 L 7 Methylcyclohexane
18 L 8 Methanol 17.5 L 9 Butylene oxide 3 L 10 Toluene 11 L 11
Butylene oxide 7 L 12 Toluene 10 L 13 Methylcyclohexane 7 L 14
Methanol 13 L 15 Methanol 14 L .sup.3The used material was a salt
mixture containing 48% bromide and 52% chloride according to the
CoA of the supplier. Thus, the real mass was different from the
theoretical one as depicted on the formula scheme.
Procedure
[0177] 1. A 100 L reactor was evacuated and purged with nitrogen
twice. [0178] 2. The 100 L reactor was charged with Compound A (1)
and Compound D (2) at JT=20.degree. C. [0179] 3. Butylene oxide (3)
and toluene (4) were charged to the reactor at JT=20.degree. C. The
100 L reactor was evacuated and purged with nitrogen twice. The
reaction mixture was warmed to JT=100.degree. C. A homogenous
solution was obtained. [0180] 4. The solution was stirred at
JT=100.degree. C. for 4 h. (IT was about 98.degree. C.). [0181] 5.
The solution was concentrated under a slight vacuum at
JT=110.degree. C. 9.0 L solvent was removed. The mixture was
stirred at JT=110.degree. C. for 13 h. (IT was about 105.degree.
C.). [0182] 6. The mixture was diluted with butylene oxide (5) and
cooled to IT=20.degree. C. (2.75 h). A red suspension was formed.
[0183] 7. A sample for an IPC was taken.
TABLE-US-00011 [0183] IPC1#1 No aldehyde signals were detected
(.sup.1H-NMR), see Note 1.
[0184] 9. The suspension was filtered on a filter dryer within 100
min. [0185] 10. Cold ethanol (6) was used to rinse the 100 L
reactor. The rinse solution was transferred to the filter dryer.
[0186] 11. The filter cake was washed with methylcyclohexane (7).
[0187] 12. The filter cake was dried on a rotary evaporator for 4.5
h. at 55.degree. C. [0188] 13. 5.246 kg crude product was obtained
as a red solid. An .sup.1H-NMR spectrum revealed the presence of a
substantial quantity of TPPO (about 25%-30%) in addition to the
desired product. [0189] 14. The crude product was transferred on a
filter dryer and washed with methanol (8). [0190] 15. The filter
cake was dried on a rotary evaporator for 19 h. at 55.degree. C.
[0191] 16. 3.787 kg Compound E was obtained as a red solid
(crude3#1). The identity was confirmed by H-NMR, The purity was
93.70% a/a as measured by HPL. In addition, 3.17+2.54% cis isomers
were also observed. The yield (nc) was 54.1%. [0192] 17. The mother
liquor (about 81 L) was concentrated under a slight vacuum at
JT=100.degree. C. 30 L solvent was removed. The mixture was stirred
at JT=110.degree. C. for 12.5 h. (IT was about 105.degree. C.).
[0193] 18. The mixture was diluted with butylene oxide (9) and
toluene (10) and cooled to IT=20.degree. C. (2.75 h). A red
suspension was formed. [0194] 19. A sample for an IPC was taken,
cooled to 0.degree. C. and filtered.
TABLE-US-00012 [0194] IPC3#1 Still too much TPPO in product
(.sup.1H-NMR).
[0195] 20. The mixture was diluted with butylene oxide (11) and
toluene (12), warmed to JT=60.degree. C. and cooled again to
IT=20.degree. C. [0196] 21. A filtered sample was taken and washed
with methanol.
TABLE-US-00013 [0196] IPC3#2 Content of TPPO in product was reduced
significantly (.sup.1H-NMR).
[0197] 22. The suspension was cooled to 1.degree. C. within 60 min
and filtered on a filter dryer within 60 min. [0198] 23. The filter
cake was washed with methylcyclohexane (13). [0199] 24. The filter
cake was washed twice with methanol (14+15). [0200] 25. The filter
cake was dried on a rotary evaporator for 4.5 h. at 55.degree. C.
[0201] 26.705 g crude product was obtained as a red solid
(crude2#1). The identity was confirmed by .sup.1H-NMR. The purity
was 90.01% a/a as measured by HPLC. Additionally, 3.83%+5.34% cis
isomers were noted on the HPLC. The yield (nc) was 10.1%. The total
corrected yield was 63.0%.
Notes
[0202] 1) The ratio of isomers was about 60:40 trans/cis according
to the HPLC.
Sample Preparation
TABLE-US-00014 [0203] In-Process-Control Conversion: About 0.5 ml
of the reaction (IPC): mixture were taken, evaporated and analysed
by .sup.1H-NMR or HPLC. Purity: 6-7 mg of the product were analysed
by HPLC; method HPLC-TSC-M1.1. .sup.1H-NMR's 5-10 mg of the product
were dissolved in 0.9 ml CDCl.sub.3 (internal standard: TMS) for
NMR spectroscopy.
Synthesis of TSC from Compound E
TABLE-US-00015 ##STR00017## ##STR00018## Size: 3.70 kg Compound A
96.2 mol (nc) Th. Quantity: 3.58 kg TSC Pr. Quantity: 2.19 kg TSC
5.88 mol (nc) Yield (nc): 6.12% Purity: 98.76% a/a HPLC Molecular
Pos. Reagent Wt. Eq. Quantity Unit 1 Compound E 384.51 1.0 3.70 kg
2 Ethanol 15.7 L 3 30% NaOH 15.0 L 4 Water 45 L 5 Ethanol 3.5 L 6
Water 39 L 7 Water 39 L 8 Water 38 L 9 Ethanol 39 L
Procedure
[0204] 1. A 100 L reactor was evacuated and purged twice with
nitrogen. [0205] 2. The 100 L reactor was charged with Compound E
(1) at JT=20.degree. C. [0206] 3. Ethanol (2) and 30% NaOH (3) were
charged to the 100 L reactor at JT=20.degree. C. The 100 L reactor
was evacuated and purged with nitrogen twice. The reaction mixture
was warmed to JT=90.degree. C. A thick, orange suspension was
obtained. [0207] 4. The suspension was stirred at JT=90.degree. C.
for 63 h (IT at 81.degree. C.). [0208] 5. The mixture was cooled to
IT=21.degree. C. within 2 h. A sample for an IPC was taken.
TABLE-US-00016 [0208] IPC1#1 98.7% conversion monoester to TSC
(HPLC); no diester (Compound E) was detected.
[0209] 6. The mixture was diluted with water (4). [0210] 7. The
suspension was filtered on a filter dryer within 15 h. [0211] 8.
Ethanol (5) was used to rinse the 100 L reactor. [0212] 9. The
filter cake was washed three times with cold water (6, 7+8),
between 0.degree. C.-5.degree. C. [0213] 10. The filter cake was
washed with ethanol (9). [0214] 11. The filter cake was dried on a
rotary evaporator for 5 h. at 50.degree. C. [0215] 12. 2.186 kg TSC
was obtained as an orange solid. The identity was confirmed by
.sup.1H-NMR. The purity was 97.96% a/a as measured by HPLC. The
yield (nc) was 61.2%; see Note 1. The water content was determined
to be 1.58% w/w and the UV ratio 421 nm to 254 nm to be 8.9. Anal
Calc for C.sub.20H.sub.22O.sub.4Na.sub.2-0.34H.sub.2O: C, 63.47; H,
6.04; Na, 12.15; 0, 18.35. Found: C, 63.81; H, 5.64; Na, 12.21; 0,
18.34. [0216] 13. 2.184 kg crude1#1 was shaken in a blender for 4
days at RT. The remaining lumps were easily ground down with a
pestle. 2.183 kg crude2#1 (corresponded to AA-013329-Batch-01-2004)
orange solid was obtained. The purity was 98.76% a/a HPLC. A DSC
measurement revealed no difference.
Notes
[0216] [0217] 1) The product is soluble in water. Thus, the long
filtration time as well as the additional water washing might have
caused the low yield observed in this specific situation. The third
water washing was performed to fit the desired sodium content in
product.
Sample Preparation
TABLE-US-00017 [0218] In-Process-Control Conversion: About 0.5 ml
of the reaction (IPC): mixture were taken and analysed by HPLC.
Purity: 6-7 mg of the product were analysed by HPLC; method
HPLC-TSC-M1.2. .sup.1H-NMR's 5-10 mg of the product were dissolved
in 0.9 ml D.sub.2O for NMR spectroscopy.
Analytics
HPLC Methods
[0219] Method: HPLC-TSC-M. 1.2 [0220] Replaces Method:
HPLC-TSC-M.1.1 [0221] Method valid for: C-009594, Compound A, TSC,
Compound D, Compound E [0222] Chemicals: Acetonitrile, HPLC grade
(J. T. Baker or equivalent) Water, HPLC grade (Milli-Q system
purified or equivalent) Trifluoroacetic acid (Merck or equivalent)
THF, HPLC grade, without stabilizer (Scharlau or equivalent) MeOH,
HPLC grade (Scharlau or equivalent) [0223] Equipment:
HP-1100-System or equivalent [0224] Column: YMC Pack Pro C18,
100.times.4.6 mm, 3 .mu.m
Mobile Phase Preparation:
Solution A: 0.1% TFA in H.sub.2O/ACN 90:10% v/v
Solution B CAN
Sample Preparation:
Mixing Solvents THF, MeOH, H.sub.2O, ACN
[0224] [0225] C-009594, C-013327. 6-7 mg (accurately weighed) of
the crude material is dissolved in MeOH [0226] C-0146796-7 mg
(accurately weighed) of the crude material is dissolved in 30 mL
ACN and filed up with water to the 100 mL line. [0227] 6-7 mg
(accurately weighed) of the crude material is dissolved in 100 mL
THF. [0228] C-014681: 6-7 mg (accurately weighed) of the crude
material is dissolved in 10 mL H.sub.2O and [0229] C-013329: filled
up to 100 mL with THF.
HPLC Parameters:
TABLE-US-00018 [0230] Column: YMC Pack Pro C18, 100 .times. 4.6 mm,
3 .mu.m Mobile A: 0.1% TFA in H.sub.2O/ACN 90:10% v/v phase: B: ACN
Gradient: 0.00 min 30% B 12.00 min 95% B 16.00 min 95% B 16.10 min
30% B 20.00 min 30% B Flow rate: 1 mL/min Temperature: 20.degree.
C. Detection: 421 nm (if detector is able to: 220, 230, 254 and 350
nm in addition) Injection 10 .mu.L volume:
Integration Parameters:
NA
[0231] Identification Table for the Following HPLC Traces:
TABLE-US-00019 Retention molecular relative times* weight Proposed
structures/ retention [min] [g/mol] C- numbers times 3.54 469
Compound D 0.51 4.47 164 Compound A 0.65 4.99 278 C-009594 0.72
6.90 372 TSC 1.00 14.30 384 Compound E 2.07 *typical retention
times for G-1172 with column LC-0402.
UV Methods
[0232] Modified method for this synthesis: [0233] Method:
UV-TSC-M.2.1 [0234] Replaces Method: - [0235] Method valid for: TSC
[0236] Chemicals: Water, HPLC grade (Milli-Q system purified or
equivalent) [0237] Equipment: Perkin Elmer Lambda 25-System or
equivalent
[0238] Cuvette: 1 cm Quartz glass [0239] UV parameters: Wavelength:
421 nm; 350 nm; 254 nm [0240] Sample preparation: 10 mg (accurately
weighted) was dissolved in 50 mL water. The sample was
ultrasonicated for 30 min. at 450-50.degree. C. 1 mL of this
solution was diluted with an additional 49 mL of water (making a
total solution volume of 50 ml). [0241] Document and method
history: Sample preparation of customer method modified in order to
measure within the linear range.
Example 3
Synthesis of Trans Potassium Crocetinate (di-potassium
2,6,11,15-tetramethylhexadeca-2E,4E,6E,8E,10E,12E,14E,-heptane-1,16-diota-
te)
[0242] Trans potassium crocetinate is also referred to below as TPC
or Compound F. The
##STR00019##
[0243] The saponification reaction (last step in the synthesis) was
performed in a similar fashion to the process used in Examples 1
and 2 above. The diethyl ester, Compound E, was prepared as
described above in Examples 1 and 2.
[0244] For this example, Compound E was treated with 30% potassium
hydroxide (KOH) (1.5 ml/mmol) in ethanol (EtOH) (1.5 ml/mmol) at
90.degree. C. for 4 days. The mixture was diluted with ethanol due
to some solvent loss during the process. The orange product was
isolated by filtration at room temperature (22.degree. C.) and by
washing with 50% ethanol in water (3.times.). The product was dried
on a rotary evaporator for 5 h at JT=60.degree. C. This produced a
reaction yield of 86% (12 g experiment). An .sup.1H-NMR spectrum
and an LC-MS spectrum confirmed the desired product was trans
potassium crocetinate. The HPLC quality was 98.3% using the HPLC
method described above in Example 2 with a detection wavelength of
421 nm.
Example 4
Synthesis of Trans Lithium Crocetinate (di-lithium
2,6,11,15-tetramethylhexadeca-2E,4E,6E,8E,10E,12E,14E,-heptane-1,16-diota-
te)
[0245] Trans lithium crocetinate is also referred to below as TLC
or Compound G. The chemical synthesis is shown below.
##STR00020##
[0246] The saponification reaction was performed by a process
similar to that outlined in Examples 1-3. In this case, however,
lithium hydroxide was used as the saponification agent. In this
example, Compound E was again synthesized as described in Examples
1 and 2. Compound E was treated with 10% lithium hydroxide (LiOH)
(2.8 ml/mmol) in ethanol (EtOH) (1.5 ml/mmol) at 90.degree. C. for
4 days. The dark-orange suspension was filtered at room temperature
(22.degree. C.) and was washed with 50% ethanol in water (3.times.)
and pure ethanol. The dark orange solid was dried on a rotary
evaporator for 5 h at jacket temperature (JT)=60.degree. C. This
produced 9.5 g dark-orange solid.
[0247] HPLC analysis of this compound indicated 38% a/a starting
material remained in addition to the desired product. The
substantial amount of remaining starting material is likely a
result of using a less concentrated basic solution (LiOH) in this
saponification reaction. In this case, given the limited solubility
of LiOH in water, only a 10% solution of LiOH in water was used. In
the other examples (1-3 described herein), a 30% basic solution in
water was used.
[0248] In order to improve the reaction yield, the isolated product
from the previous step was treated with solid LiOH (13 eq.) in 50%
EtOH in water (2.9 ml/mmol) at 95.degree. C. for further 2 days.
The orange product resulting from this second treatment with
lithium hydroxide was isolated by filtration at room temperature
and by washing with 50% EtOH in water (3.times.) and pure EtOH. The
resulting solid was dried on a rotary evaporator for 3 h at jacket
temperature=60.degree. C. This gave a yield of 14 g product.
Because this was more than the theoretical yield, an additionally
slurry in water (1.6 ml/mmol) was performed to remove the excess of
LiOH. The orange product was isolated by filtration at room
temperature and washing with 50% EtOH in water (3.times.) and pure
EtOH. The product was dried on a rotary evaporator for 2 h at
jacket temperature=60.degree. C. This gave a yield of 8.5 g, which
corresponds to an overall reaction yield of 67% (12 g
experiment).
[0249] Both .sup.1H-NMR and LC-MS spectra confirmed the desired
product, trans lithium crocetinate, was obtained. The HPLC quality
was 99.7% using the analysis procedure described above in Example 1
with a detection wavelength set at 421 nm.
Example 5
Synthesis of the C-14 Derivative of TSC (disodium
4,9-dimethyldodeca-2E,4E,6E,8E,10E-pentane-1,12 diotate)
[0250] The synthesis of symmetric compounds with a shorter chain
length than TSC required the use of a different Wittig agent other
than the Compound B shown in Examples 1-4. Shorter chain length
BPTC compounds are synthesized using Compound A, the C-10
dialdehyde used previously. Compound A was then converted via
either a Horner-Emmons reaction or a Wittig coupling reaction with
either a commercially-available C2 or C3-phosphonate or a
phosphonium bromide (Compound H) to form the corresponding C14- and
C16-esters, respectively. Hydrolysis with NaOH/EtOH completed the
reaction, resulting in the formation of the desired C14 or
C16-derivatives of TSC.
[0251] The synthesis of the C-14 derivative of TSC, Compound J, was
completed via the reaction of Compound A and Compound H
(ethoxycarbonyl-methyl-triphenyl-phosphonium bromide). Compounds A
and H reacted to form the final intermediate, Compound I, prior to
a saponification step which produced Compound J as shown below.
##STR00021##
Coupling Reaction to Form Compound I
[0252] The best method for the formation of Compound I consisted of
using the C2 phosphonium bromide (Compound H). This coupling
reaction was performed in butylene oxide/toluene 2:1 (0.7 ml+1.4
ml/mmol) as used in Examples 1-4 above. The result was good product
formation with 3 eq. Wittig reagent at jacket
temperature=100.degree. C. A yellow product was isolated by
filtration at 0.degree. C. and subsequent washing (twice) with
methylcyclohexane. The yield for this step Was 56-61% (10 g
scale).
[0253] The purity was determined to be 83.4% a/a trans isomer at a
detection wavelength of 369 nm (11.5 min) using HPLC. In addition
to Compound I, an additional three compounds were observed to be
present in the HPLC trace and are speculated to be cis isomers of
Compound I (1.0% a/a, 10.5 min; 13.4% a/a, 11.2 min; 2.0% a/a, 11.9
min).
Saponification Reaction to Form the Final Product (Compound J)
[0254] The saponification reaction to convert compound I to
Compound J was performed in a manner similar to that described in
Examples 1-4. The diethyl ester, Compound I, was treated with 30%
NaOH (2 ml/mmol) in EtOH (4 ml/mmol) at 90.degree. C. for 3 days.
The yellow product was isolated by filtration at room temperature
and by washing with 50% EtOH in water (5.times.) and pure EtOH
(3.times.). The saponification reaction gave a crude yield of about
100%. In order to improve the purity, the mixture was slurried in
30% NaOH (0.5 ml/mmol) in EtOH (1 ml/mmol) at 90.degree. C. for 4
h. The suspension was filtered at 0.degree. C. and washed with EtOH
(3.times.) giving a yellow product, which corresponded to an
uncorrected yield of 79% (10 g scale). .sup.1H-NMR confirmed that
the desired product, Compound J, was synthesized with an HPLC
quality of 97.8% with a detection wavelength set at 383 nm.
Example 6
Synthesis of the C-15 Derivative of TSC (disodium
2,4,9-Trimethyldodeca-2E,4E,6E,8E,10Epentaene-1,12-diotate)
[0255] Compound A was reacted with Compound K
(1-(Ethoxycarbonyl)-ethyltriphenylphosphoniumbromide), a C3
phosphonium salt/C3 Wittig ester bromide to produce the first
intermediate in this reaction sequence, Compound M. Alternatively,
in this first step, the same result can be achieved if Compound A
is reacted with Compound L (Triethyl-2-phosphonopropionate), a C3
phosphono ester, to produce intermediate Compound M.
[0256] In the second coupling reaction, Compound M was reacted with
Compound H (ethoxycarbonyl-methyltriphenylphosphoniumbromide), a C2
phosphonium salt/C2 Wittig ester bromide, to form the penultimate
intermediate, Compound O. Alternatively, Compound O can be formed
via a reaction between Compound M and Compound N
(triethyl-phosphono-acetate), a C2 phosphono ester.
[0257] In the final reaction step, Compound O underwent a
saponification reaction to form the C-15, asymmetrical derivative
of TSC, Compound P. The reaction scheme is shown in the information
below.
##STR00022##
[0258] The C15 derivative required the use of two different mono
coupling reactions in sequence; the first one with the C3 phosphono
ester or phosphonium bromide, and the second with the C2
phosphonium bromide. Due to the formation of some C16 diethyl
ester, the crude product from the first monocoupling reaction was
purified by a chromatography over silica gel.
First Coupling Reaction to Form Compound M
[0259] Compound A was treated with 1 eq. C-3 phosphonium bromide
(Compound K) at 100.degree. C. for 1 day. This coupling reaction
was performed in butylene oxide/toluene 2:1 (0.7 ml+1.4 ml/mmol)
and showed good product formation. While cooling to room
temperature and later to 0.degree. C., no precipitation was
observed. The orange mixture was then filtered over silica gel
(0.33 g/mmol). The filter cake was washed with toluene (1.7
ml/mmol). The filtrates were combined and evaporated to dryness at
JT=45.degree. C. to give a crude product that appeared as an orange
oil.
[0260] Next, the reaction mixture was evaporated to dryness. The
residue was slurried in MeOH to obtain the crude product in solid
form and then washed with MeOH (3.times.). The quality of the crude
product obtained was poor due to the presence of C16 diethylester
in addition to the desired C13 mono coupling product, Compound M.
Thus, a purification step over silica gel with 10:1
methylcyclohexane/EtOAc was performed. The crude product was
dissolved in dichloromethane (0.3 ml/g crude product). The C-16
diethyl ester (about 7%) was the first compound to elute. Once this
step was performed, the yield was 45% (20 g experiment). On a
smaller-scale, yields were as high as 70%. The best quality product
was determined to have 98.1% a/a trans isomer at a detection
wavelength of 383 nm (9.22 min). An additional compound was present
(1.7% a/a, 9.47 min) which was suspected to be a cis isomer of
Compound M.
Second Coupling Reaction to Form Compound O
[0261] Compound M was treated with 1.5 eq C-2 phosphonium bromide
(Compound H) at 100.degree. C. This coupling reaction was performed
in butylene oxide/toluene 2:1 (0.5 ml+1.0 ml/mmol) and showed a
good product formation. A yellow product was isolated by filtration
at 0.degree. C. and by washing twice with methylcyclohexane. The
yield at this step was 36%.
[0262] In an effort to improve the yield, the mother liquor was
concentrated to about half of the original volume and cooled to
0.degree. C. to obtain a second crop with a yield of 27%. The total
yield of both crops combined was 63% (13 g scale).
[0263] The best quality product (Compound O) was determined to have
87.0% a/a trans isomer at a detection wavelength of 383 nm (12.4
min). In addition, a second compound was noted on the HPLC trace
(11.3% a/a, 11.8 min) and might be a cis isomer of Compound O.
Saponification Reaction to Form the Final Product (Compound P)
[0264] The diethyl ester, Compound O, was treated with 30% NaOH (2
ml/mmol) in EtOH (4 ml/mmol) at 90.degree. C. for 3 days. A
greenish, yellow product was isolated by filtration at RT and
washing with 50% EtOH in water (5.times.) and EtOH (3.times.). The
saponification reaction produced a yield of 83% (6 g scale).
[0265] An .sup.1H-NMR experiment confirmed the desired product,
Compound P. The HPLC quality was 97.0% Compound P at a detection
wavelength of 383 nm. In addition, other possible cis isomers
corresponding to 1.4% a/a+1.2% a/a were noted. LCMS-data confirmed
the given structure.
Example 7
Synthesis of the C-16 Derivative of TSC (disodium
2,4,9,11-Tetramethyldodedeca-2E,4E,6E,8E,10E-pentaene-1,12-diotate)
[0266] Compound A was reacted with Compound L
(Triethyl-2-phosphonopropionate), a C3 phosphono ester, to form the
critical intermediate in this synthesis, Compound Q.
[0267] Alternatively, Compound Q can be produced by a reaction
between Compound A and Compound K
(1-(Ethoxycarbonyl)-ethyltriphenylphosphoniumbromide), a C3
phosphonium salt/C3 Wittig ester bromide. Although the initial
reactants (Compounds A, K and L) are the same as found in Example
6, the reaction conditions used here yielded a different, symmetric
intermediate, Compound Q.
[0268] In the final step of the reaction, Compound Q was reacted
with sodium hydroxide and ethanol in a saponification reaction that
produced the final product, Compound R. Compound R (disodium
2,4,9,11-tetramethyldodedeca-2E,4E,6E,8E,10E-pentaene-1,12-diotate)
is the C-16, symmetric derivative of TSC. The reaction scheme is
shown in the information below.
##STR00023##
Coupling Reaction to Form Compound Q (using Compound L as a
reactant)
[0269] The synthesis of the C16-derivative (Compound R) was
commenced in a fashion similar to that shown in Example 6--a
coupling reaction between Compound A and Compound L. Compound A was
treated with Compound L at 100.degree. C. in toluene/butylene oxide
2:1. No chemical reaction was observed without the addition of a
base and in this case, the mixture was cooled to 0.degree. C. and
NaOMe (3 eq.) was added.
[0270] A second quantity of reagent, prepared from C-3 phosphono
ester (2.times.1.5 eq.) and NaOMe in DCM twice, was added to the
reaction mixture. The mixture was stirred at JT=65.degree. C. and a
higher percentage of desired products were observed. HPLC traces
indicated that only a small percentage of unreacted Compound A was
remaining (2.3% a/a). The reaction mixture was quenched with water
and the organic phase was washed in a solution consisting of:
water, a saturated NaHCO.sub.3 solution and a 50% saturated. NaCl
solution. The result was the production of 36.4 g of Compound Q
after evaporation.
[0271] Purification of Compound Q was undertaken using silica gel
10:1 methylcyclohexane/EtOAc. The result was 0.26 g light yellow
product (Compound Q) and 4.0 g of a yellow solid (Compound M). The
transesterification product, Compound Q, (dimethylester instead of
the diethylester) was isolated with a yield of 32% using MeOH as a
solvent. An alcohol solvent would have been suitable for the
coupling reaction as well. Overall, the quality of product from
this step was excellent at 85.7% purity. HPLC experiments showed
the majority of the impurities were cis isomers of Compound Q.
Coupling Reaction to Form Compound Q (Using Compound K as a
Reactant)
[0272] In a second experiment to synthesize Compound Q, Compound K
was substituted for compound L in the coupling reaction. This
substitution showed a better overall reaction conversion. The
coupling reaction between Compound A and Compound K was performed
in a mixture of butylene oxide/toluene 2:1 (0.9 ml+1.8 ml/mmol) and
showed good product formation with 3 eq. Wittig reagent at
JT=100.degree. C. (22 h). The yellow product was isolated by
filtration at 0.degree. C. and by washing twice with
methylcyclohexane. The yield was 61-62% (10 g scale). HPLC analysis
showed a purity of Compound Q at 85.2% a/a trans isomer at 369 nm
(13.3 min) with the major impurity being a cis isomer.
Saponification Reaction to Form the Final Product (Compound R)
[0273] The saponification reaction to produce Compound R was
conducted in a manner similar to that described in Example 5. The
diethyl ester (Compound Q) was treated with 30% NaOH (2 ml/mmol) in
EtOH (4 ml/mmol) at 90.degree. C. for 3 days. The saponification
gave a yield of 85% (12.5 g scale) after filtration at RT and
washing with 50% EtOH in water (3.times.) and EtOH (3.times.). The
.sup.1H-NMR confirmed the desired product was Compound R. The HPLC
quality was 95.7% with a detection wavelength of 383 nm.
Additionally, 3.8% cis isomer was observed. LCMS-data confirmed the
given structure.
Example 8
Synthesis of the C-17 Derivative of TSC (disodium
2,6,11-Trimethyltetradeca-2E,4E,6E,8E,10E,12Ehexaene-1,14-diotate)
[0274] The preparation of a longer-chain, asymmetric derivative of
TSC was performed using Compounds A and D as starting materials as
in Examples 1-2. However, in this case, the reaction conditions
were changed to favor the formation of the first coupling
intermediate, Compound S. Compound S was then reacted in a second
coupling reaction with Compound H to form Compound T. In the final
step of this reaction sequence, Compound T underwent a
saponification reaction with sodium hydroxide and ethanol to form
the final, desired product, Compound U. Compound U is the C-17
derivative of TSC and asymmetric in the placement of the pendant
methyl groups around the diene backbone. Details of the synthetic
sequence are shown in the information below.
##STR00024##
First Coupling Reaction to Form Compound S
[0275] Compound A was treated with 1 eq. Compound D at 100.degree.
C. for 1 day. This coupling reaction was performed in butylene
oxide/toluene 2:1 (0.44 ml+0.88 ml/mmol) as used in Example 1 and
showed a good product formation. During a subsequent cooling step
at 0.degree. C., a suspension was formed from the original dark red
solution. Ultimately, a red-orange solid was isolated by filtration
at 0.degree. C. and by washing with methylcyclohexane (3.times.).
This red-orange solid was identified as Compound E, previously
described in Examples 1-4. The yield of Compound E in this
situation was approximately 4%.
[0276] The mother liquor, from the previously described step, was
evaporated to dryness at JT=45.degree. C. to give the crude
Compound S product as a red oil. The quality of the obtained crude
product was poor due to the presence of some Compound E in addition
to the desired product, Compound S. In order to isolate Compound S,
a purification step over silica gel with 8:1
methylcyclohexane/EtOAc was performed. For that, the crude product
was solvated first in dicholormethane (0.3 ml/g crude product). The
first eluted product from this mixture was the C-20 diethyl ester
(Compound E). The remaining fraction contained a mixture of
compounds including the desired Compound S. The yield after this
step was 46% (30 g experiment) of an orange solid. The quality was
determined (by HPLC) to be 71.2% a/a trans isomer at a detection
wavelength of 369 nm (10.4 min). In addition, two further compounds
were identified 0% a/a+4.9% a/a) which are likely to be cis isomers
of Compound S.
Second Coupling Reaction to Form Compound T
[0277] Compound S was reacted with 1.5 eq Compound H at 100.degree.
C. This coupling reaction was performed in butylene oxide/toluene
2:1 (0.6 ml+1.2 ml/mmol) and showed a good product formation. The
red product, Compound T, was isolated by filtration at 0.degree. C.
and washing twice with methylcyclohexane. The yield was 58% (10 g
scale). The quality was determined to be 97.2% a/a trans isomer at
a detection wavelength of 421 nm (14.2 min). In addition, two other
compounds were detected and are speculated to be cis isomers of
Compound T.
Saponification Reaction to Form Compound U
[0278] The saponification reaction was performed in a similar
fashion to Examples 1-4. Compound T was treated with 30% NaOH (2
ml/mmol) in EtOH 2 ml/mmol) at 90.degree. C. for 4 days. After 2
days, the mixture was diluted with water (1 ml/mmol). The yellow
product, Compound U, was isolated by filtration at RT and washing
with 50% EtOH in water (2.times.) and EtOH (3.times.). The
saponification reaction showed a yield of 93% (7 g scale). The
.sup.1H-NMR confirmed the desired product was Compound U. The HPLC
quality was 98.4% at a detection wavelength of 399 nm with one
major impurity found at 0.7%.
Example 9
Synthesis of the C-18 Derivative of TSC (Disodium
2,4,9,13-Tetramethyltetradeca-2E,4E,6E,8E,10E,12Ehexaene-1,14-diotate)
[0279] The C18 derivative of TSC, Compound W, was prepared via two
sequential mono coupling reactions as seen in Examples 6 and 8
within this application. The first coupling reaction was commenced
as in Example 8 leading to the formation of Compound S. In the
second coupling reaction, Compound S was reacted with Compound K to
form the penultimate intermediate, Compound V. The final, desired
product--Compound W, was produced via the saponification reaction
of Compound V. The information below illustrates the details of the
synthetic sequence.
##STR00025##
First Coupling Reaction to Form Compound S
[0280] The first coupling reaction shown above, leading to the
formation of Compound S, is described previously in this
application in Example 8. The method used in this example is
identical.
Second Coupling Reaction to Form Compound V
[0281] The second coupling reaction was performed by reacting
Compound S (also known as the C15 monoester) with 1.5 eq. Compound
K (a C-3 phoshonium bromide) at 100.degree. C. This coupling
reaction was performed in butylene oxide/toluene 2:1 (0.6 ml+1.2
ml/mmol) and showed a good product formation. An orange product was
isolated by filtration at 0.degree. C. and by two washing steps in
methylcyclohexane. The yield for this step was 56% (9 g scale). The
quality of Compound V was measured using HPLC and found to be 96.7%
a/a trans isomer at a detection wavelength of 369 nm (14.2 min). In
addition, the major impurity observed (1.7% a/a, 13.6 min) is
speculated to be a cis-isomer of Compound V.
Saponification Reaction to Form Compound W
[0282] The saponification reaction was performed in a similar
fashion to the previous examples. Compound V was treated with 30%
NaOH (2.2 ml/mmol) in EtOH (3.4 ml/mmol) at 90.degree. C. for 4
days. After 2 days, the mixture was diluted with water (1.1
ml/mmol). A yellow product was isolated by filtration at RT and
washing with 50% EtOH in water (2.times.) and EtOH (3.times.). The
saponification reaction gave a yield of 98% (6 g scale). The
.sup.1H-NMR confirmed that the product obtained was the desired
product, Compound W. HPLC analysis showed the purity of Compound W
was 99.2% at a detection wavelength of 405 nm. In addition, an
impurity of 0.3% a/a/ was observed and is believed to be a cis
isomer of Compound W.
Example 10
Synthesis of the C-24 Derivative of TSC (Disodium
4,8,13,17-Tetramethyleicosa-2E,4E,6E,8E,10E,12E,14E,16E,18
Enonaene-1,10-diotate)
[0283] The longer chain derivatives of TSC required the use of
multi-step synthetic processes involving reduction and oxidation
steps. This example illustrates the synthesis of the C24 derivative
of TSC (Disodium
4,8,13,17-Tetramethyleicosa-2E,4E,6E,8E,10E,12E,14E,16E,18
Enonaene-1,10-diotate). The synthesis commenced with Compound E,
the starting point for several examples already described in this
application. Compound E (a diethyl ester) was converted to a
Compound X, a dialcohol, via a reduction step. The third step in
the reaction sequence was an oxidation of Compound X with MnO.sub.2
to form Compound Y. The next step consisted of a coupling reaction
with Compound H to form Compound Z and a final saponification step
to form the desired product, Compound AA (the C-24 derivative of
TSC). The reaction sequence is shown below.
##STR00026##
Reaction to Form Compound E
[0284] The coupling reaction between Compounds A and D to form
Compound E has been described earlier in Examples 1-4.
Reduction Reaction to Form Compound X
[0285] 40 g diethyl ester (Compound E) was suspended in toluene and
treated with DiBAI (6 eq.) at JT=-70.degree. C. The mixture was
stirred for 4.5 h. at JT=-70.degree. C. An IPC revealed complete
conversion. The mixture was quenched with 2 M HCl at -77.degree.
C.
[0286] The mixture was divided in three portions. Each portion was
diluted with water and THF. The organic layer was washed 3.times.
with brine. The combined organic layers were evaporated at
JT=45.degree. C. to give 30.63 g of an orange solid, which
corresponded to a yield of 98%. Re-extraction of the aqueous layers
gave further 1.48 g orange solid.
[0287] From the process described above, an orange product was
obtained with a quality of 89.0% a/a as measured by HPLC. In
addition, an impurity, believed to be an aldehyde side reaction
product, was observed at a concentration of 4.8% a/a isomer.
[0288] Furthermore, a separate study was undertaken to determine
whether the reduction reaction could have been performed with LAH
or NaBH.sub.4. The LAH reduction (2 eq.) in THF at 0.degree. C.
while slowly warming to RT within 4 h gave a yield of about 80% in
a 1 g experiment, but the quality was not as high as with the
DiBAI-H method. But this reagent can be an option for further
evaluations on this step. In contrast, the reduction with
NaBH.sub.4 (2 eq.) in THF was found not to be appropriate for this
step. Additionally, the addition of MeOH as a co-solvent did not
improve the reaction.
Oxidation Reaction to Form Compound Y
[0289] Two experiments were conducted in an effort to optimize the
oxidation reaction to form Compound Y. In the first experiment, the
oxidation reaction was performed using a low concentration of
Compound X (1.7%) in acetone and an excess of MnO.sub.2 (30 eq.).
The reaction was started at 0.degree. C. while warming to RT over
the course of 1 day. The reagent was removed by filtration over
celite or silica gel and evaporation to give a purple solid product
at a yield of 42-57%. The quality of product was measured by 87.7%
a/a HPLC at a detection wavelength of 421 nm. This approach led to
a high-purity product as measured by HPLC.
[0290] As a second approach to conducting the oxidation reaction, a
small-scale experiment was conducted with focus on the
crystallization procedure. The use of an acetone/water mixture at a
ratio of 2:3 (0.013 g/ml) as the solvent yielded the best results
at this step. Other solvents such as dioxane/MCH (1:2), THF and
EtOAc did not produce better crystallization results. The one
parameter that did improve crystallization was changing the jacket
temperature. Raising the jacket temperature to 75.degree. C.
significantly improved the conversion to product and reduced the
amount of MnO.sub.2 required for the overall reaction to 10 eq.
from 30 eq. A more detailed description of the steps taken in this
modified approach is described in the following paragraph.
[0291] 5 g Compound X was suspended in acetone and treated with 5
eq. MnO.sub.2 at JT=75.degree. C. The mixture was stirred for 2
days at 75.degree. C. An IPC revealed the presence of dialdehyde
and monoaldehyde species in addition to the starting material. As a
result, an additional 5 eq. MnO.sub.2 was added to drive the
reaction further towards completion. After 8 hours reaction time,
most starting material and intermediate was consumed. The mixture
was cooled and filtered first over silica gel and then subsequently
over celite. The double filtration step was necessary to remove
MnO.sub.2. A third filtration step over a membrane was conducted.
In total, washing the filter cakes with THF resulted in 0.7 g red
product besides 1.95 g+2.51 g dark brown product with traces of
manganese. The total mass yield for the reaction was 5.16 g
(99.6%). HPLC of an IPC: 61.0%+5.4%+19.3% (including isomers) at
463 nm.
[0292] In this second approach to the oxidation reaction, while the
product yield was higher than the first synthetic sequence, the
HPLC quality was not as high with residual amounts of manganese
oxide remaining in the Compound Y product.
[0293] It should be noted that in either approach, the intermediate
(monoaldehyde) must be consumed to achieve good yields and
qualities. Manganese residues can be removed via a solvent exchange
with hot THF or EtOAc prior to the filtration step.
Coupling Reaction to Form Compound Z
[0294] The synthesis of the C24 diethylester (Compound Z) was
performed in a similar fashion to Examples 5, 6 and 8 using
Compound H. The C20 dial (Compound Y) was treated with Compound H
at 100.degree. C. in toluene/butylene oxide 2:1 (1 ml and 0.5
ml/mmol) for 1 day. While cooling to RT, no precipitation was
observed. A clear, deep red solution was still present.
[0295] The mixture was then evaporated to dryness at
JT=40-50.degree. C. The residue was treated with MeOH (1.25
mmol/mmol) and cooled to 0.degree. C. The suspension was filtered
and the filter cake was washed twice with methanol. This step
resulted in a red-brown product after drying on a rotary evaporator
at JT=50.degree. C.
[0296] The yield for this reaction was 19-22%. This yield could be
improved with further reclamation of product from the mother
liquor. The best quality was determined to be 92.4% a/a trans
isomer (of Compound Z) at a detection wavelength of 421 nm (16.8
min). Additional compounds observed in the HPLC spectrum are
believed to be cis isomers of Compound Z.
Saponification Reaction to Form Compound AA
[0297] The saponification reaction was performed in a fashion
similar to those previously reported in this filing. The diethyl
ester (Compound Z) was treated with 30% NaOH (3 ml/mmol) in EtOH (3
ml/mmol) at 90.degree. C. for 3 days. The saponification reaction
produced a yield of 83% (0.8 g scale) after dilution with water (6
ml/mmol) and ethanol (3 ml/mmol), filtration at RT and washing with
50% EtOH in water (12 ml/mmol) and EtOH (12 ml/mmol). The HPLC
quality was 95.3% Compound AA at 463 nm in addition to 0.5%
cis-isomer.
Example 11
Synthesis of the C-26 Derivative of TSC (Disodium
2,4,8,13,17,19-Hexamethyl-eicosa-2E,4E
6E,8E,10E,12E,14E,16E,18Enonaene-1,10-diotate)
##STR00027##
[0298] Reaction Sequence to Form Compound Y
[0299] The reaction sequence required to form Compound Y was
previously described in Example 10. The same sequence was followed
for this example.
Coupling Reaction to Form Compound BB
[0300] The synthesis of the C26 diethylester commenced from the
production of Compound Y as described in Example 9. Compound Y was
treated with Compound K at 100.degree. C. in toluene/butylene oxide
2:1 (2 ml and 1 ml/mmol) for 1 day. While cooling to RT no
precipitation was observed. A clear, deep red solution was still
present. The mixture was cooled to 0.degree. C. The resulting
suspension was filtered. Afterwards the filter cake was washed
three times with MeOH to give a dark purple solid after drying on a
rotary evaporator at JT=45.degree. C.
[0301] The yield at this step was between 17-26%. One of the major
reasons was that some of the product remained in the mother liquor.
The best quality product was determined to have 83.9% a/a trans
isomer at a detection wavelength of 463 nm (by HPLC). Additional
compounds that were observed are speculated to be cis isomers of
Compound BB. This experiment was conducted on a 1.5 g scale.
Saponification Reaction to Form Compound CC
[0302] The saponification reaction was performed in a similar
fashion to that described in Example 9. The diethyl ester (Compound
BB) was treated with 30% NaOH (3 ml/mmol) in EtOH (3 ml/mmol) at
90.degree. C. for 3 days. The saponification reaction gave a yield
of 84% (0.8 g scale) after dilution with water (6 ml/mmol) and
ethanol (3 ml/mmol), filtration at RT and washing with 50% EtOH in
water (12 ml/mmol) and EtOH (12 ml/mmol). The .sup.1H-NMR confirmed
the desired product was obtained (Compound CC). The HPLC quality
was 91.5% at a detection wavelength of 460 nm. In addition, 0.2%
cis-isomer was observed.
Example 12
Formulation of TSC with Cyclodextrin-Mannitol
[0303] 1. Make up a solution containing equal-molar concentrations
of the cyclodextrin and the TSC. Solutions containing over 20 mg
TSC/ml of solution can be made this way. First, add the
cyclodextrinto an injectable water, then add the TSC to that
solution. [0304] 2. Add d-mannitol, so that the final concentration
is around 20 to 50 mg/ml of mannitol in solution. [0305] 3. This
solution can be added to isotonic saline in order to dilute it and
still maintain the proper osmolality. Either add the solution to
saline or add saline to the solution.
Example 13
Formulation of TSC with Mannitol/Acetic Acid
[0305] [0306] 1. Make up a 0.01 M acetic acid solution in distilled
water. [0307] 2. Combine this solution with an injectable water in
proper proportions so as to have a final acetic acid concentration
of 0.0005 M. Note: Do not use much stronger acetic acid. For
example, 0.0006 M is okay, but 0.001 M doesn't dissolve the TSC.
[0308] 3. Add the 0.0005 M acetic acid solution, slowly, to TSC.
Maximum solubility is around 6 to 6.5 mg TSC/ml solution. [0309] 4.
Add d-mannitol to above solution at a concentration of 50 mg/ml in
order to obtain the proper osmolality. The pH of this solution is
around 8 to 8.5. It should be close to being an isotonic solution,
and, as such, can be injected directly into the blood stream.
Example 14
Pulmonary Administration
[0310] TSC has been shown, in rats, to be absorbed into the blood
stream following pulmonary administration. In this method, the rats
were intubated and a small volume (0.1 mL usually) of the TSC
solution was injected followed by two 3-mL puffs of air. It was
found that 40 to 70% of the dosage given was rapidly present in the
blood stream (time to reach the maximum plasma concentration was
less then 5 minutes).
[0311] Additional studies have been done with pulmonary dosing in
which the effect of the volume of liquid injected into the trachea
as well as using TSC in its formulated drug product form were
investigated. The formulated drug product contained 8%
.gamma.-cyclodextrin, 2.3% mannitol and 50 mM glycine and 20 mg/mL
TSC, reconstituted in sterile water. Sterile saline (0.9%) was
added as a diluent in these studies in order to achieve the desired
dosages. The same dose (937 .mu.g/kg) was administered to all
rats.
[0312] It was found that the incorporation of the
.gamma.-cyclodextrin appears to enhance absorption of TSC into the
systemic circulation--with the overall effect of increasing plasma
clearance. Also, an increase in the injection volume results in
greater TSC absorption and over a longer period of time. Thus, a
larger volume injection of the same dose results in a greater
bioavailability. It should also be noted that it has been found
that hemorrhagic shock in rats can be successfully treated by
administering TSC via the pulmonary route.
Example 15
Intramuscular Administration
[0313] TSC is not absorbed via an intramuscular route when simply
dissolved in de-ionized water; however, the addition of a
cyclodextrin (as in the formulated drug product) results in
absorption into the blood stream. Small volumes (0.05 mL) were
injected into each thigh muscle of rats with TSC formulated with
2-hydroxypropyl-1-cyclodextrin or .gamma.-cyclodextrin and
dissolved in water. Intramuscular administration of 3347 .mu.g/kg
body weight (with 14 mg 2-hydroxypropyl-.beta.-cyclodextrin per kg
body weight) resulted in a peak plasma TSC concentration of 4.8
.mu.g/mL and a bioavailability of 0.27. Administration of
.gamma.-cyclodextrin with TSC also resulted in successful
absorption into the systemic circulation. Hemorrhagic shock in rats
was successfully treated by administering TSC via intramuscular
injection.
Example 16
Transdermal Administration
[0314] TSC has been shown, in rats, to be absorbed into the blood
stream following transdermal administration. For these studies,
select areas around the abdomen and/or outer thigh was either cut
and/or shaved to expose the stratum corneum. The formulated drug
product (8% .gamma.-cyclodextrin, 2.3% mannitol and 50 mM glycine
and 20 mg/mL TSC) was applied to the exposed stratum corneum and
0.25 to 0.5% of the dosage given was present in the blood stream at
a time of 15 to 30 minutes after being given.
Example 17
Oral Administration
[0315] PE-50 tubing was used to deliver formulated TSC into the
stomachs of rats, and plasma concentrations were measured after
that. Rats were fasted for 24 hours prior to each experiment. Water
was given ad libitum and coprophagy was prevented by using cages
with wire-mesh floors. One study involved rats that were allowed
food ad libitum after TSC administration and another study involved
rats in which food was withheld after TSC administration. The dose
of TSC given in both groups was 55 mg/kg and it was found that 1 to
2% of the dosage was present in the blood stream at a time of 15 to
30 minutes after being given.
Example 18
Endotoxin Removal of Gamma-Cyclodextrin
[0316] Commercially available pharmaceutical grade gamma
cyclodextrin obtained from the manufacturer has endotoxin levels
that are incompatible with intravenous injection. The endotoxin
levels must be reduced in order to use the gamma cyclodextrin in a
TSC formulation intended for intravenous injection. A process
utilizing multiple filtration passes of a cyclodextrin solution
through an endotoxin removal filter (Millipore 0.22 micron Durapore
filter) was developed that reduces endotoxin levels about 10 to 30
fold. The recovery of cyclodextrin is 90-100%. An example of the
results obtained by using this process on an 8% gamma cyclodextrin
solution is in the table below.
TABLE-US-00020 Endotoxin levels (EU/mg Filtration Steps of
cyclodextrin) prefiltered 0.226 After 1 filtration 0.0246 After 2
filtrations 0.0125 After 3 filtrations 0.0125
Example 19
Lyophilization
[0317] Lyophilization process to produce a cake of less than 3%
moisture is as follows:
TABLE-US-00021 Step Temp (degrees C.) Pressure Time (hours) Loading
ambient n/a n/a Freezing -30 n/a 1 Freezing -30 n/a 6 Evacuation
-30 225 um n/a Drying -30 225 um 2 Drying 30 225 um 15 Drying 30
225 um 4 Drying 30 50 um 99
Diluent
[0318] A diluent of glycine buffer (e.g. 50 mM glycine buffer with
2-4% mannitol) with osmolality adjusted with mannitol can be used
as a diluent.
Example 20
Radiation Sensitization
[0319] HCT116 human colon carcinoma tumors measuring between 0.25
and 0.35 cm.sup.3 were grown on the hind legs of athymic male mice
(6-7 weeks at purchase), which required 2-3 weeks growth before
use. Because patients are not normally anesthetized during
radiotherapy (anesthesia may decrease blood flow to the tumor and
make it more hypoxic), the study was performed with
non-anesthetized animals.
[0320] There were six study groups of five mice per group (for a
total of 30 mice) designated Study Groups 1 through 6. All mice in
the study were injected intravenously with either a TSC dose or a
saline control for five successive days. As shown below, Study
Groups 1, 2 and 3 received TSC doses A, B and C, which corresponds
to 0.07, 0.14 and 0.28 mg/kg respectively for the study B, and
1.35, 0.54 and 0.18 mg/kg respectively for the study A.
TABLE-US-00022 Designation N (Athymic Male Mice) Dose Radiotherapy
Group 1 Five (5) TSC Dose A Yes Group 2 Five (5) TSC Dose B Yes
Group 3 Five (5) TSC Dose C Yes Group 4 Five (5) Saline Control Yes
Group 5 Five (5) TSC Dose B No Group 6 Five (5) Saline Control
No
[0321] Group 4 was injected as a saline control. At 45 minutes
post-injection on each of the five successive days, the tumors of
Study Groups 1-4 received 2 Gy irradiation. Study Group 5 received
TSC only and Study Group 6 received saline only, and neither Study
Group 5 nor 6 received irradiation. Tumor volumes in all Study
Groups were measured weekly for the earlier of 4 weeks or until the
tumors reached 4 times the volume at the start of treatment. The
results from these tests are shown in FIGS. 1 and 2.
[0322] The optimal dosage for the Study A was 0.18 mg/kg and for
the Study B, the dosages of 0.07 and 0.14 mg/kg worked equally
well. The use of TSC alone was also studied. Those results are
shown in FIG. 3, along with the effect of radiation alone (which is
also shown in FIG. 2). It can be seen that TSC alone does not
appreciably affect tumor growth.
[0323] It will be readily apparent to those skilled in the art that
numerous modifications and additions can be made to both the
present compounds and compositions, and the related methods without
departing from the invention disclosed.
* * * * *