U.S. patent application number 14/647237 was filed with the patent office on 2015-10-15 for bryoid compositions, methods of making and use thereof.
This patent application is currently assigned to APHIOS CORPORATION. The applicant listed for this patent is APHIOS CORPORATION. Invention is credited to Trevor Percival Castor.
Application Number | 20150291616 14/647237 |
Document ID | / |
Family ID | 50828594 |
Filed Date | 2015-10-15 |
United States Patent
Application |
20150291616 |
Kind Code |
A1 |
Castor; Trevor Percival |
October 15, 2015 |
BRYOID COMPOSITIONS, METHODS OF MAKING AND USE THEREOF
Abstract
Embodiments of the present invention feature novel Bryoid
compositions, methods of making and methods of treating disease
Inventors: |
Castor; Trevor Percival;
(Arlington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APHIOS CORPORATION |
Woburn |
MA |
US |
|
|
Assignee: |
APHIOS CORPORATION
Woburn
MA
|
Family ID: |
50828594 |
Appl. No.: |
14/647237 |
Filed: |
November 26, 2013 |
PCT Filed: |
November 26, 2013 |
PCT NO: |
PCT/US13/72070 |
371 Date: |
May 26, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61730227 |
Nov 27, 2012 |
|
|
|
Current U.S.
Class: |
514/450 ;
549/348 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/365 20130101; A61P 31/12 20180101; A61P 31/18 20180101;
A61P 25/14 20180101; C07D 493/22 20130101; A61P 27/06 20180101;
A61P 31/22 20180101; A61P 25/00 20180101; A61P 25/28 20180101; A61P
25/16 20180101 |
International
Class: |
C07D 493/22 20060101
C07D493/22 |
Goverment Interests
STATEMENT REGARDING FEDERAL SPONSORSHIP
[0002] The inventions of the present application were developed
with Federal sponsorship under National Institute on Aging and
National Institutes of Health Grant Number 5R44AG034760.
Claims
1. A first Bryoid composition having a molecular weight of
approximately 896-898 Amu (Mass+Sodium) and 873-875 Amu
(monoisotopic mass) having a purity of approximately 50% to a
crystal forming purity.
2. The first Bryoid composition of claim 1 having a measured mass
plus sodium of 897.2 Amu and a measured monoisotopic mass of 874.2
Amu.
3. A second Bryoid composition having a molecular weight of
approximately 910-912 Amu (Mass+Sodium) and 888-890 Amu
(monoisotopic mass) having a purity of approximately 50% to a
crystal forming purity.
4. The second Bryoid composition of claim 3 having a measured mass
plus sodium of 911.5 Amu and a measured monoisotopic mass of 888.9
Amu.
5. A third Bryoid composition having a molecular weight of
approximately 868-870 Amu (Mass+Sodium) and 846-848 Amu
(monoisotopic mass) having a purity of approximately 50% to a
crystal forming purity.
6. The third Bryoid composition of claim 5 having a measured mass
plus sodium of 869.5 Amu and a measured monoisotopic mass of 846.6
Amu.
7. A fourth Bryoid composition having a molecular weight of
approximately 895-897 Amu (Mass+Sodium) and 872-874 Amu
(monoisotopic mass) having a purity of approximately 50% to a
crystal forming purity.
8. The fourth Bryoid composition of claim 7 having a measured mass
plus sodium of 895.5 Amu and a measured monoisotopic mass of 872.6
Amu.
9. A method of making a Bryoid composition comprising the steps
isolating a Bryoid composition from a source of Bryoids and
purifying the Bryoid composition to a purity of 50% and a crystal
forming purity said Bryoid composition selected from the group of
Bryoids consisting of the first Bryoid, second Bryoid, third Bryoid
and fourth Bryoid.
10. A method of treating a disease responsive to Bryoid
compositions comprising the step of administering an effective
amount of a bryoid composition selected from the group of Bryoids
consisting of the first Bryoid, second Bryoid, third Bryoid and
fourth Bryoid.
Description
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/730,227, filed Nov. 27, 2012, the entire
contents of which are incorporated herein by reference.
FIELD OF INVENTION
[0003] Embodiments of the present invention are directed to
compositions having utility as therapeutics in neurodegenerative
diseases such as Hutchinson Disease, Parkinson's disease, Down's
syndrome and Alzheimer's disease, virus latency diseases such as
HIV and Herpes, cancers such as prostate and other amyloid mediated
diseases such as glaucoma.
BACKGROUND OF THE INVENTION
[0004] Neurodegenerative diseases, such as Alzheimer's disease,
Hutchinson's Disease, Parkinson's disease, Kuru, Creutzfeldt-Jakob
disease and other spongiform encephalopathies remain major health
problems. Currently there are very limited means to treat these
diseases. With respect to Alzheimer's, Hutchinson's and Parkinson's
diseases, these diseases tend to manifest themselves in older
individuals and as the diseases progress; the afflicted individuals
are less able to care for themselves. The neurogenerative diseases
are associated with the formation of beta amyloid plaques.
Bryostatin 1 stimulates the production of certain isoforms of
protein kinase C (PKC) that increase the production of
alpha-secretase which makes soluble amyloid precursor protein, thus
inhibiting the formation of beta amyloid plaques, With respect to
cancers such as prostate cancer, Bryostatin 1 inhibits phorbol
ester-induced apoptosis in prostate cancer cells by differentially
modulating protein kinase C (PKC) delta translocation and
preventing PKCdelta-mediated release of tumor necrosis
factor-alpha. With respect to virus latency diseases such as HIV
latency, Bryostatin-1, as well as many PKC agonists, activates
cellular transcription factors such as NF-kB that binds the HIV-1
promoter and regulates its transcriptional activity. In HIV-1
latency the viral promoter is less accessible to cellular
transcription factors because nuclear histones surrounding the
viral promoter are deacetylated (compacted chromatin). Thus HDAC
inhibitors may increase the aceytation of histones (relaxed
chromatin) and then transcription factors may have an easy access
to the HIV promoter.
[0005] Bryoids consist of a family of bryostatins that are complex
cyclic macrolide molecules. Bryoids were originally isolated from
the marine bryozoan, Bulgula neritina, in small quantities. Methods
of synthesis are awkward and costly. About twenty Bryoid
compositions, known as bryostatins and numbered 1-20, have been
identified. Many of the bryoids are known to possess anti-cancer
properties.
[0006] It would be useful to have new Bryoid compounds that possess
high potency and activity.
SUMMARY OF THE INVENTION
[0007] Embodiments of the present invention feature a first Bryoid
composition having a molecular weight of approximately 896-898 Amu
(Mass+Sodium) having a purity of approximately 50% to a crystal
forming purity. The first Bryoid composition can also be
characterized as a Bryoid compound having a molecular weight of
approximately 873-875 Amu (monoisotopic mass) having a purity of
approximately 50% and a crystal forming purity. The first Bryoid
composition has a measured mass plus sodium of 897.2 Amu and a
measured monoisotopic mass of 874.2 Amu. The detailed discussion
which follows will refer to this Bryoid as B10.
[0008] Embodiments of the present invention feature a second Bryoid
composition having a molecular weight of approximately 910-912 Amu
(Mass+Sodium) having a purity of approximately 50% to a crystal
forming purity. The second Bryoid composition can also be
characterized as a Bryoid compound having a molecular weight of
approximately 888-890 Amu (monoisotopic mass) having a purity of
approximately 50% and a crystal forming purity. The second Bryoid
composition has a measured mass plus sodium of 911.5 Amu and a
measured monoisotopic mass of 888.9 Amu. The detailed discussion
which follows will refer to this Bryoid as B12.
[0009] Embodiments of the present invention feature a third Bryoid
composition having a molecular weight of approximately 868-870 Amu
(Mass+Sodium) having a purity of approximately 50% to a crystal
forming purity. The third Bryoid composition can also be
characterized as a Bryoid compound having a molecular weight of
approximately 846-848 Amu (monoisotopic mass) having a purity of
approximately 50% and a crystal forming purity. The third Bryoid
composition has a measured mass plus sodium of 869.5 Amu and a
measured monoisotopic mass of 846.6 Amu. The detailed discussion
which follows will refer to this Bryoid as B14B.
[0010] Embodiments of the present invention feature a fourth Bryoid
composition having a molecular weight of approximately 895-897 Amu
(Mass+Sodium) having a purity of approximately 50% to a crystal
forming purity. The fourth Bryoid composition can also be
characterized as a Bryoid compound having a molecular weight of
approximately 872-874 Amu (monoisotopic mass) having a purity of
approximately 50% and a crystal forming purity. The fourth Bryoid
composition has a measured mass plus sodium of 895.5 Amu and a
measured monoisotopic mass of 872.6 Amu. The detailed discussion
which follows will refer to this Bryoid as B14C.
[0011] These Bryoid compounds of the present invention have
molecular weights that are different than the molecular weights of
bryostatins 1-20.
[0012] As used herein, crystal forming purity means the composition
has a purity which enables the composition to form crystals.
Normally, such purity is greater than 90%, and more often greater
than 95% purity. Examples presented in this application feature
compositions having a purity greater than 99%. Crystal purity would
comprise compositions in which no impurities can be detected, but
is not so limited.
[0013] The Bryoid composition of the present invention has utility
in the treatment of Bryoid responsive conditions such as
neurodegenerative diseases, cancers and virus latencies. The Bryoid
composition of the present invention is highly active modulators of
certain isoforms of protein kinase C (PKC) and amyloid precursor
protein. The Bryoids, and the Bryoid composition of the present
invention, stimulate the production of certain isoforms of protein
kinase C (PKC) that increase the production of alpha (alpha)
secretase which transforms amyloid precursor protein into soluble
forms. Bryoids composition of the present invention exhibit high
levels of activity similar to or greater than bryostatin 1.
[0014] One embodiment of the present invention is directed to the
treatment of a disease such as a neurodegenerative disease, cancer
and virus latency responsive to Bryoids, such as Bryostatins 1-20.
The method comprises the step of administering an effective amount
of at least one bryoid composition selected from the group
consisting of the first Bryoid composition, the second Bryoid
composition, the third Bryoid composition, and the fourth Bryoid
composition.
[0015] Embodiments of the present invention further comprise the
Bryoid composition selected from the group consisting of the first
Bryoid composition, the second Bryoid composition, the third Bryoid
composition, and the fourth Bryoid composition in a dosage form for
administration to a patient. The dosage form may take many forms
including without limitation, intravenous, intraperitoneal, oral
dosage forms, such as tablets, gel caps, capsules, oral solutions
and suspensions; aerosols, such as spray or mist forming solutions
for administration to lungs, or nasal passageways, topical forms
such as ointments, lotions, patches and sprays; and other dosage
forms known in the art.
[0016] A further embodiment of the present invention is directed to
a method of making a Bryoid composition selected from the group
consisting of the first Bryoid composition, the second Bryoid
composition, the third Bryoid, and the fourth Bryoid composition
comprising the steps of isolating the a Bryoid composition from a
source of Bryoids and purifying the Bryoid composition to a purity
of 50% and a crystal forming purity. The source of Bryoids is
preferably the marine bryozoan, Bugula neritina.
[0017] These and other features and advantages of the present
invention will be apparent upon viewing the Figures and reading the
detailed descriptions that follow.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 depicts a high performance liquid chromatography scan
of Bryostatin 1;
[0019] FIG. 2 depicts a HPLC Chromatogram of B. neritina Ethyl
Acetate (EA) crude extract;
[0020] FIG. 3 depicts a flow chart of purifying steps for
Bryostatin-type compositions;
[0021] FIG. 4 depicts alpha-secretase activity induced by
Bryostatin-1 and other extracts which embody aspects of the present
invention;
[0022] FIG. 5 depicts a chromatogram of a mixture of Bryoids;
[0023] FIG. 6 depicts a chromatogram of a mixture of Bryoids and
identifies retention times monitored at 265 nm;
[0024] FIG. 7 depicts UV spectra of different Bryoids at 265
nm;
[0025] FIG. 8-1 depicts a mass spectrum of Bryostatin 1, scanning
from 700-1000 Amu;
[0026] FIG. 8-2 depicts a mass spectrum of a fraction with an
internal identifications 104 and B08 associated with Bryostatin 2,
scanning from 700-1000 Amu;
[0027] FIG. 8-3 depicts a mass spectrum of a fraction with internal
designations 106 and B14 associated with Bryostatin 3, scanning
from 700-1000 Amu;
[0028] FIG. 8-4 depicts a mass spectrum of a fraction with internal
designations 112 and B16 embodying features of the present
invention, scanning from 700-1000 Amu;
[0029] FIG. 8-5 depicts a mass spectrum of a fraction with internal
designations 102 and B12 and B14 associated with Bryostatin-3,
scanning from 700-1000 Amu;
[0030] FIG. 8-6 depicts a mass spectrum of a fraction with internal
designations 103 and B10 and B12, scanning from 700-1000 Amu;
[0031] FIG. 8-7 depicts a mass spectrum of a fraction with internal
designations 105 and B12, and B14 associated with Bryostatin-3
scanning from 700-1000 Amu;
[0032] FIG. 9 depicts UV spectra of the first Bryoid composition
and the second Bryoid composition;
[0033] FIG. 10 depicts the effect of Bryostatin-1 and different
Bryoids at 10-.sup.9M on alpha-secretase activity in SHSY-5Y
neuroblastoma cells;
[0034] FIG. 11 depicts the effect of Bryostatin-1 and different
Bryoids at 10-.sup.9M on PKC-epsilon activity in SHSY-5Y
neuroblastoma cells;
[0035] FIG. 12 depicts the effect of Bryostatin-1 and different
Bryoids at 10-.sup.9M on PKC-delta activity in SHSY-5Y
neuroblastoma cells;
[0036] FIG. 13 depicts the effect of Bryostatin-1 and different
Bryoids at 10-.sup.9M on PKC-alpha activity in SHSY-5Y
neuroblastoma cells;
[0037] FIG. 14 depicts the proposed structure of a first
Bryoid;
[0038] FIG. 15 depicts the proposed structure of a second
Bryoid;
[0039] FIG. 16 depicts the NMR spectra of Bryostatin-3;
[0040] FIG. 17 depicts the NMR spectra of the first Bryoid;
and,
[0041] FIG. 18 depicts the NMR spectra of the second Bryoid.
DETAILED DESCRIPTION OF THE INVENTION
[0042] Embodiments of the present invention will now be described
with respect to a Bryoid composition selected from the group
consisting of the first Bryoid composition (sometimes referred to
as B10), the second Bryoid composition (sometimes referred to as
B12), the third Bryoid composition (sometimes referred to as B14B),
the fourth Bryoid composition (sometimes referred to as B14C).
These Bryoid compounds of the present invention have molecular
weights that are different than the molecular weights of
Bryostatins 1-20, with the exception of B12 which appears to be a
stereoisomer of Bryostatin 3.
[0043] Bugula neritina was fractionated to produce Bryostatin
fractions (Bryoids) and isolate individual Bryoids.
HPLC Analysis:
[0044] Bryostatin-1 was analyzed by HPLC using a 15 cm 5 micron
Phenomenex Luna PFP (2) column (UPS Packing L43) and a mobile phase
of 60% acetonitrile acidified with 50 microliters of 85%
H.sub.3PO.sub.4 per liter. The flow rate was set to 1.0 mL per
minute and the column temperature was set at 30.degree. C. A Waters
Millennium system incorporating a Model 996 photodiode array
detector was used to generate the chromatographic scans (FIG. 1).
Bryostatin-1 was monitored at 265 nm, and contour plots were
simultaneously reported from 195 nm to 345 nm.
Bryostatin-1 Manufacturing and Characterization:
[0045] In the first two steps, Bryostatins are extracted from wet
Bugula neritina with organic solvents including isopropanol,
methanol, ethyl acetate and water followed by silica chromatography
using mobile phases consisting of hexane/methylene chloride and
ethyl acetate/methanol or alternatively extracted from washed,
dried and milled Bugula neritina with SuperFluids.TM.
(near-critical and supercritical fluids with or without cosolvents)
carbon dioxide and methanol and partially purified by
SuperFluids.TM. silica chromatography with carbon dioxide and
methanol (Castor, 1998, 2001).
[0046] The third step is a segmentation chromatography step on a
CG71 polymeric resin (Rohm-Haas) with a mobile phase consisting of
methanol and water that improves the purity of Bryostatin-1 to
60-70%. The fourth step utilizes a segmentation chromatographic
method using two semi-prep HPLC C18 columns (Baker Scientific,
Phenomenex) with a mobile phase consisting of acetonitrile and
water to improve the Bryostatin-1 purity to >95%. The fifth step
utilizes crystallization with acetonitrile and water to purify
Bryostatin-1 to >98.5%.
[0047] The identity of the Bryostatin-1 product was confirmed by
Ultra Violet (UV) spectra as well as High Performance Liquid
Chromatography (HPLC) retention times versus those of standards
provided by the U.S. National Cancer Institute (NCI), National
Institutes of Health (NIH), Bethesda, Md. The identity of the
Bryostatin-1 product was also confirmed independently by Mass
Spectral (MS) data as well as by Elemental Analysis, Proton and
Carbon Nuclear Magnetic Resonance (NMR), Infra Red (IR)
spectroscopy, Differential Scanning calorimetry (DSC) and Melting
Point.
Purification of Bryostatin-1 to 99.64% CP
Purification Procedure and Results:
[0048] An ethyl acetate extract of B. neritina (Sample C-021519#7),
provided by the National Cancer Institute (NCI), was used as the
starting raw materials. A total of .about.57 g of the EA extract
was dissolved in dichloromethane (DCM) and assayed to determine
presence of Bryostatin-1 and other Bryoids. Turning now to FIG. 2,
a HPLC Chromatogram of B. neritina EA crude extract is depicted.
Labeled arrows indicate internal designations for Bryostatin-like
compounds, which include B10, B12, and Bryostatin 3 (B16), and
Bryostatin-2 (Bryo-2) and Bryostatin-3 (Bryo-3). Bryostatin-1
(Bryo-1) elutes at 24.2 min. The designation B10 is the bryoid
corresponding to the first bryoid of the present invention. The
designation of B14 will lead to the third and fourth Bryoids of the
present invention.
[0049] Bryostatin-1 was purified from B. neritina EA crude extracts
using various chromatography resins as shown in FIG. 3. The initial
steps (Step 1 and Step 2) were performed on Silica gel Active
(100-200 .mu.m), and the sample eluted with increasing
concentrations of ethyl acetate in DCM. The silica purification
steps are useful in removing some of the colored components from
the EA crude extract, the non-polar compounds (eluting at end of
chromatographic run, FIG. 3), and eliminating the majority of the
B16 peak.
[0050] Next, fractions containing Bryostatin-1 were purified on
Amberchrom CG71, which allowed for the elution of Bryostatin-like
compounds with acidified methanol and water. This resin helps
minimize the use of chlorinated solvents that are harmful to the
environment. CG71 purification step removes the `X5` peak eluting
before Bryostatin-1. It also served to minimize the impurities
right before Bryostatin-1, mainly B16.
[0051] Subsequent purification was performed using a combination of
Amicon C18 40 .mu.m resin and two prep-C18 columns (2.5.times.2.5
cm, 10 .mu.m column) This step allowed for the further separation
of B12 and Bryo-3 from Bryostatin-1, though there was still the
presence of the `x5` peak at the shoulder of Bryostatin-1. Final
crystallization step led to the purification of Bryostatin-1 to
>99% chromatography purity (CP), with a 69% recovery from crude
extract.
HPLC Monitoring:
[0052] During each purification step outlined in FIG. 3,
Bryostatin-1 was monitored on a Luna C18(2) column (250.times.4.6
mm, 10 .mu.m). Elution was performed at 80% acetonitrile acidified
with phosphoric acid (ACNP) in an isocratic mode at a 2 mL/min flow
rate. Column temperature was set at 30.degree. C.
Bryostatin-Like Compounds (Bryoids)
[0053] Bugula neritina was fractionated to produce Bryostatin
fractions (Bryoids) that could serve as alternatives to
Bryostatin-1. These fractions were purified and sent to LSU for in
vitro analysis (Table 1).
TABLE-US-00001 TABLE 1 Amount (in mg) of each Bryoid in the
Fractions as determined by HPLC.sup.1 Fraction Sample B08 B10 B12
B14 Bryo-1 B16 % CP A: 101 B157 88.8 97.5 165 mL B: 102 B154 30.5
101.1 4.2 74.4 C: 103 B158 B12 60.4 100.8 6.7 49.3 350 mL D: 104
Bryo-2 100.9 94.4 E: 105 Bryo-AB 99.8 53.7 64.5 F: 106 Bryo-3 98.3
12.1 72.1 G: 112 B16 APH 99.5 97.5 100311 CP corresponds to Bryoid
in bold
Efficacy of Bryostatin-1 Analogues (Bryoids) in Induction of
s-APP.alpha. Secretion:
[0054] The efficacy of several Bryostatin-1 analogues (Bryoids) in
induction of s-APP.alpha. secretion is shown in FIG. 4. Except
Fraction D, they all induced significant release of s-APP.alpha.
compared to Bryostatin-1. The best alternative fraction to
Bryostatin-1 is analogue E which corresponds to the designation
B16, which was identified as Bryostatin 3. Bioactivity went in
order from Fraction E (105)>G (112), F (106), C (103)>A
(101), B (102)>D (104).
[0055] From the preliminary data, it appears that B12 or B14 can be
significantly more bioactive than Bryostatin-1. Since most
fractions contain two or more Bryoids, it is difficult to determine
which one is responsible for the bioactivity except for Fraction G,
which contains B16 at >97.5% CP. The first bryoid composition of
the present invention, B10, has significantly higher activity than
Bryostatin-1, and it poses another potential alternative Bryoid as
a therapeutic.
HPLC Standardization:
[0056] Turning now to FIG. 5, which depicts a high performance
liquid chromatograph of a bryoid mixture at 265 nm, various Bryoids
are present in the B. neritina EA crude extract.
[0057] The mixture of the Bryoids (B09, B10, B12, B14C, B14B, B16,
Bryostatin-1, Bryostatin-2, and Bryostatin-3) were standardized for
the purpose of identification (based on retention time) and
subsequent purification of each Bryoid. A chromatogram depicting
the results of high performance liquid chromatography purification
is depicted in FIG. 6. These Bryoids contain similar UV patterns as
Bryostatin-1 with a maximum wavelength at 265 nm as shown in FIG. 7
which depicts UV spectra of different bryoid at 265 nm.
[0058] Identification of each Bryoid was attempted on UV-HPLC and
LC/MS/MS using known standards and/or comparing to known masses in
the literature. This is important as previous preliminary in vitro
experiments (described above) have shown that these Bryoids may
induce s-APP.alpha. secretion at equal or even greater percentages
than is observed for Bryostatin-1.
Preliminary Characterization Based on LC/MS/MS
[0059] Characterization of the different Bryoids was performed
using an LC/MS/MS API 2000 system equipped with a Shimadzu HPLC
system. Q1 scan parameters were optimized for Bryostatin-1 m/z 427
[M+Na] (FIG. 8-1), scanning from 700 to 1000 amu. Mass spectrum
scans of other fractions are presented in FIGS. 8-2 through 8-7. A
total of seven fractions were analyzed, which included individual
Bryoids and mixture of Bryoids (Table 2).
TABLE-US-00002 TABLE 2 Bryostatin Analogue for Each Fraction Based
on Mass Match Bryostatin Bryoid Mass + Na Mass [M] Match Based on
Mass Fraction 101: Bryo-1 927.3 904.3 Bryostatin-1 Fraction 102:
B12 and 911.4 888.4 Bryostatin-3 B14 (Bryo-3) 925.4 902.4 None
Fraction 103: B10 and 911.4 888.4 Bryostatin-3 B12 897.2 874.2 None
Fraction 104: Bryo-2 885.4 862.4 Bryostatin-2 Fraction 105: B12 and
911.4 888.4 Bryostatin-3 Bryo-3 Fraction 106: Bryo-3 911.4 888.4
Bryostatin-3 Fraction 112: B16 909.4 886.4 None(tentatively
identified as Bryostatin-3)
Discussion:
[0060] The LC/MS/MS data observed for Bryostatin-1 shows a peak at
927 Amu, which corresponds to the [M+NA], and what has been
reported in the literature (Manning et al., 2005). Mass spectral
data on Bryostatin-1 to 18 are summarized in Table 3. Based on the
LC/MS/MS analysis performed, Fractions 104 and Fraction 106 were
confirmed as Bryostatin-2 (863 Amu) and Bryostatin-3 (889 Amu),
receptively.
[0061] Fraction 112 showed that B16 mass does not match any Bryoids
reported in the literature. Fractions 102, 103, and 105 showed a
mass peak identical to what was observed for Bryostatin-3. Both
Fraction 102 and 105 contain Bryo-3 in their mixture, which would
explain the 911 peak observed in the LC/MS/MS. It is unclear why
911 Amu is seen in Fraction 103; this indicates that B12 may have
the same mass as Bryostatin-3 (889 Amu). This is supported by the
fact that Fraction 105, containing both B12 and Bryo-3, only showed
a peak at 911 Amu. The 897 peak observed in Fraction 103 could
correspond to B10, though it does not match any of the Bryostatin
masses reported in the literature. The peak at 925 Amu in Fraction
102 is also observed in Fraction 106.
TABLE-US-00003 TABLE 3 Mass Spectral Information on Bryostatin-1 to
18 (Manning et al., 2005) Monoisotopic M. M. + (Na+): M. M. .+-.
(H.sub.2): Group R1 monoisotopic Group R2 monoisotopic Empirical
Bryo. # mass 22.9892 2.0156 mass (attached) mass (attached) formula
1 904.4456 927.4348 902.4300 59.0133: 139.0759:
C.sub.47H.sub.68O.sub.17 906.4613 CH.sub.3COO
CH.sub.3(CH.sub.2).sub.2(CH).sub.4COO 2 862.4350 885.4243 860.4194
17.0027: 139.0759: C.sub.45H.sub.66O.sub.16 864.4507 OH
CH.sub.3(CH.sub.2).sub.2(CH).sub.4COO 3 888.4143 911.4035 886.3987
59.0133: 139.0759: C.sub.46H.sub.64O.sub.17 890.4300 CH.sub.3COO
CH.sub.3(CH.sub.2).sub.2(CH).sub.4COO 4 894.4613 917.4505 892.4456
101.0602: 87.0446: C.sub.46H.sub.70O.sub.17 896.4769
(CH.sub.3).sub.2CHCH.sub.2COO CH.sub.3(CH.sub.2).sub.2COO 5
866.4300 889.4192 864.4143 101.0602: 59.0133:
C.sub.44H.sub.66O.sub.17 868.4456 (CH.sub.3).sub.2CHCH.sub.2COO
CH.sub.3COO 6 852.4143 875.4035 850.3987 87.0446: 59.0133:
C.sub.43H.sub.64O.sub.17 854.4300 CH.sub.3(CH.sub.2).sub.2COO
CH.sub.3COO 7 824.3830 847.3722 822.3674 59.0133: 59.0133:
C.sub.41H.sub.60O.sub.17 826.3987 CH.sub.3COO CH.sub.3COO 8
880.4456 903.4348 878.4300 87.0446: 87.0446:
C.sub.45H.sub.68O.sub.17 882.4613 CH.sub.3(CH.sub.2).sub.2COO
CH.sub.3(CH.sub.2).sub.2COO 9 852.4143 875.4035 850.3987 87.0446:
59.0133: C.sub.43H.sub.64O.sub.17 854.4300
CH.sub.3(CH.sub.2).sub.2COO CH.sub.3COO 10 808.4245 831.4137
806.4088 101.0602: 1.0078: C.sub.42H.sub.64O.sub.15 810.4401
(CH.sub.3).sub.3CCOO H 11 766.3775 789.3667 764.3619 59.0133:
1.0078: C.sub.39H.sub.58O.sub.15 768.3932 CH.sub.3COO H 12 932.4769
955.4661 930.4613 87.0446: 139.0759: C.sub.49H.sub.72O.sub.17
934.4926 CH.sub.3(CH.sub.2).sub.2COO
CH.sub.3(CH.sub.2).sub.2(CH).sub.4COO 13 794.4088 817.3980 792.3932
87.0446: 1.0078: C.sub.41H.sub.62O.sub.15 796.4245
CH.sub.3(CH.sub.2).sub.2COO H 14 824.4194 847.4086 822.4037
101.0602: 17.0027: C.sub.42H.sub.64O.sub.16 826.4350
(CH.sub.3).sub.3CCOO OH 15 920.4405 943.4297 918.4249 59.0133:
155.0708: C.sub.47H.sub.68O.sub.18 922.4562 CH.sub.3COO
CH.sub.3CH.sub.2CHOH(CH).sub.4 COO 16 790.4139 813.4031 788.3983
101.0602: 1.0078: C.sub.42H.sub.62O.sub.14 792.4296
(CH.sub.3).sub.3CCOO H 17 790.4139 813.4031 788.3983 101.0602:
1.0078: C.sub.42H.sub.62O.sub.14 792.4296 (CH.sub.3).sub.3CCOO H 18
808.4245 831.4137 806.4088 101.0602: 1.0078:
C.sub.42H.sub.64O.sub.15 810.4401 (CH.sub.3).sub.3CCOO H
Isolation of Bryostatin Analogues: B16 (98.5% CP) and B14B (93.4%
CP)
[0062] Bryoid-like compounds, B16 and B14B, were purified from
side-cuts collected from previous Bryostatin-1 purifications, and
had been stored at 4.degree. C. The bryoids' UV-spectra are
identical to that of Bryostatin-1 (FIG. 9).
HPLC Monitoring:
[0063] During purification, B16 and B14B were monitored on a Luna
C18(2) column (250.times.4.6 mm, 10 .mu.m). Elution was performed
at 80% ACNP (acetonitrile acidified with phosphoric acid) isocratic
mode, at a 2 mL/min flow rate. Column temperature was set at
30.degree. C.
Purification Procedure and Results:
[0064] Fractions containing B16 and B14B were purified using two
prep-C18 columns (2.5.times.2.5 cm, 10 .mu.m) and a semi-prep PFP
column Purification was performed with step-gradient using
increasing concentrations of ACNP. Elution was monitored until each
Bryoid was located mainly on individual columns. Columns were
stripped using a fast gradient with ACNP, and fractions were
assayed to determine concentration of each peak.
[0065] Bryoids B16 and B14 B can be separated successfully using
the described column system. The use of both C18 and PFP column is
necessary for the separation of B16 from B14B, and partial
purification of B14B from B14C. Peak labeled B14C is another bryoid
that co-elutes with B14B, and can be better monitored when analyzed
at 70% ACNP. Crystallization of both B16 and B14B/C was possible by
addition of MeOH to the Bryoid-containing fractions. A total of 212
mg of B16 crystals with 98.5% CP were collected. A total of 108 mg
of B14B/C at 93.4% CP was recovered and stored for future
purification. B14B/C was subsequently separated into B14B and B14C.
The purified Bryoids were re-analyzed by LC/MS/MS. The results are
summarized in Table 4.
TABLE-US-00004 TABLE 4 LC/MS/MS Analysis of Purified Bryostatin
Analogues for Each Fraction Based on Mass Match Bryoid Mass + Na
Mass [M] Bryostatin Match Based on Mass Bryostatin-1 927.3 904.3
Bryostatin-1 Bryostatin-2 885.4 862.4 Bryostatin-2 Bryostatin-3
911.4 888.4 Bryostatin-3 B16 909.4 886.4 None B10 897.4 874.4 None
B12 911.5 888.9 Bryostatin-3 Isomer B14B 869.5 846.6 None B14C
895.5 872.6 None
Biological Activities of Purified Bryoids:
[0066] Purified Bryoids at 10-.sup.9M are shown to increase
alpha-secretase activity in SHSY-5Y neuroblastoma cells in FIG. 10.
B3, B14B and B16 are shown to improve the production of
alpha-secretase over Bryostatin-1.
[0067] B10 is shown to improve the production of PKC-epsilon over
Bryostatin-1 in FIG. 11. B10 is shown to improve the production of
PKC-delta over Bryostatin-1 in FIG. 12. B10 is shown to improve the
production of PKC-alpha over Bryostatin-1 in FIG. 13.
NMR and Structural Characterization:
[0068] The three variants were compared by to bryostatin 1 and
bryostatin 3 by their NMR .sup.1H and .sup.13C resonances and
connectivities (HSQC and HMBC spectra). All three variants
distinctly had the ring closure at C22 of bryostatin 3, and similar
R1 and R3 sidechains (the OAc and the 8-carbon 2,4-ene). The
variations, relative to bryostatin 3, were:
[0069] B10: NMR showed loss of one methyl group from C18, matching
the mass difference: Predicted C.sub.45H.sub.62O.sub.17=874.4
(monoisotopic); obs B10 874.4. The putative structure of B10 is
depicted in FIG. 14.
[0070] B12 appears to be a stereoisomer: a number of protons in the
vicinity of the 19-24 ring have modest changes in chemical shift;
but the connectivities show the same covalent structure as
bryostatin 3, and it has the same mass as bryostatin 3
(C.sub.46H.sub.64O.sub.17=888.4). The most likely site would be at
C22, if the mechanism of ring closure was not perfectly
stereoselective. Inversion at adjacent sites (19, 20, or 23) could
also explain the NMR changes, although these variations are not
seen among the other bryostatins. The putative structure for B12 is
depicted in FIG. 15.
[0071] In B16, the 26-OH has become a ketone. This change accounts
for the 2 Da observed mass difference between B16
(C.sub.46H.sub.62O.sub.17=886.4) and Bryo-3 (888.4). A bryostatin-3
26-ketone is known (Schaufelberger 1991).
[0072] These structures are suggested by the NMR data which is set
forth in NMR spectra in FIGS. 16-18. FIG. 16 depicts the NMR
spectra of Bryostatin-3. FIG. 17 depicts the NMR spectra of B10.
FIG. 18 depicts the NMR spectra of B12 overlaid on the NMR spectra
of Bryostatin-3.
[0073] Thus, we have disclosed embodiments of the present invention
based on our present understanding of the best mode to make and use
these compounds. Those skilled in the art will readily understand
that such preferred embodiments are subject to alteration and
modification and therefore the present invention should not be
limited to the precise details, but should encompass the subject
matter of the claims that follow and their equivalents.
* * * * *