U.S. patent application number 11/825092 was filed with the patent office on 2007-11-22 for byrostatin analogues, synthetic methods and uses.
This patent application is currently assigned to The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Kevin W. Hinkle, Blaise Lippa, Cheol-Min Park, Paul A. Wender.
Application Number | 20070270485 11/825092 |
Document ID | / |
Family ID | 29554126 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070270485 |
Kind Code |
A1 |
Wender; Paul A. ; et
al. |
November 22, 2007 |
Byrostatin analogues, synthetic methods and uses
Abstract
Biologically active compounds related to the bryostatin family
of compounds, having simplified spacer domains and/or improved
recognition domains are disclosed, including methods of preparing
and utilizing the same.
Inventors: |
Wender; Paul A.; (Menlo
Park, CA) ; Lippa; Blaise; (Stonington, CT) ;
Park; Cheol-Min; (Gurnee, IL) ; Hinkle; Kevin W.;
(Chapel Hill, NC) |
Correspondence
Address: |
KING & SPALDING LLP
1180 PEACHTREE STREET
ATLANTA
GA
30309-3521
US
|
Assignee: |
The Board of Trustees of the Leland
Stanford Junior University
|
Family ID: |
29554126 |
Appl. No.: |
11/825092 |
Filed: |
July 3, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10366776 |
Feb 14, 2003 |
7256286 |
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11825092 |
Jul 3, 2007 |
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09728929 |
Nov 30, 2000 |
6624189 |
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11825092 |
Jul 3, 2007 |
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60357177 |
Feb 15, 2002 |
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60168181 |
Nov 30, 1999 |
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Current U.S.
Class: |
514/450 ;
514/452; 514/460; 549/267; 549/416; 549/427 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 309/06 20130101; C07D 493/08 20130101; C07F 7/1804 20130101;
C07D 309/10 20130101; C07D 319/16 20130101; C07D 319/14 20130101;
C07D 493/22 20130101; C07D 407/06 20130101 |
Class at
Publication: |
514/450 ;
514/452; 514/460; 549/267; 549/416; 549/427 |
International
Class: |
A61K 31/335 20060101
A61K031/335; A61K 31/35 20060101 A61K031/35; A61P 35/00 20060101
A61P035/00; C07D 315/00 20060101 C07D315/00; C07D 321/00 20060101
C07D321/00 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This invention was made with the support of NIH grant number
CA31845. Accordingly, the U.S. Government may have certain rights
in this invention.
Claims
1. A compound having the structure represented by a formula of the
group: ##STR130## ##STR131## wherein: R.sup.3 is H, OH or a
protecting group; R.sup.6 is H, H or .dbd.O; R.sup.8 is H, OH,
.dbd.O, R', --(CH.sub.2).sub.nO(O)CR' or
(CH.sub.2).sub.nCO.sub.2-haloalkyl where n is 0, 1, 2, 3, 4 or 5,
provided that R.sup.6 and R.sup.8 are not both .dbd.O; R.sup.9 is
H, OH or is absent; R.sup.20 is H, OH, or -T-U--V--R' where: T is
--O--, --S--, --N(H)-- or --N(Me)--; U is absent or is --C(O)--,
--C(S)--, --S(O)-- or --S(O).sub.2--; and V is absent or is --O--,
--S--, --N(H)-- or --N(Me)--, provided that V is absent when U is
absent; R.sup.21 is .dbd.CR.sup.aR.sup.b or R.sup.21 represents
independent moieties R.sup.c and R.sup.d where: R.sup.a and R.sup.b
are independently H, CO.sub.2R', CONR.sup.cR.sup.d or R'; R.sup.c
and R.sup.d are independently H, alkyl, alkenyl, alkynyl or
(CH.sub.2).sub.pCO.sub.2R' where p is 1, 2 or 3; R' is
independently selected from: H, alkyl, alkenyl, alkynyl, aryl,
heteroaryl, aralkyl and heteroaralkyl; R'' is OH, OTBS or OBn; and
X is --CH.sub.2--, --O--, --S-- or --N(R.sup.e)-- where R' is COH,
CO.sub.2R' or SO.sub.2R', or a pharmaceutically acceptable salt
thereof.
2. A compound of claim 1 having one or more of the stereochemical
configurations represented by the corresponding formula of the
group: ##STR132## ##STR133##
3. The compound or salt of any of claims 1 to 3 where: R.sup.8 is
H, alkyl, aralkyl, or --O.sub.2C-lower alkyl, provided that for
Formula C26 des-methyl 705 R.sup.8 is other than
--O.sub.2C-t-butyl; R.sup.9 is H; R.sup.20 is H, OH,
--O.sub.2C-lower alkyl or --O.sub.2C-alkenyl; R.sup.21 is
.dbd.C--CO.sub.2-lower alkyl; and X is --CH.sub.2-- or --O--.
4. The compound or salt of claim 3 where: R.sup.3 is OH; R.sup.8 is
H, t-butyl, --O.sub.2C--CH.sub.3, --O.sub.2C--C(CH.sub.3).sub.3 or
--O.sub.2C--CH.sub.2--CH.sub.2--CH.sub.3; and R.sup.20 is H, OH,
--O.sub.2C--CH.sub.3, --O.sub.2C--CH.sub.2--CH.sub.2--CH.sub.3 or
--O.sub.2C--CH.dbd.CH--CH.dbd.CH--CH.sub.2--CH.sub.2--CH.sub.3.
5. A compound having the structure represented by a formula of the
group: ##STR134## ##STR135## wherein: R.sup.7 is absent or
represents from 1 to 4 substituents on the ring to which it is
attached, independently selected from: lower alkyl, hydroxyl,
amino, alkoxyl, alkylamino, .dbd.O, acylamino and acyloxy; R.sup.20
is H, OH, or -T-U--V--R' where: T is --O--, --S--, --N(H)-- or
--N(Me)--; U is absent or is --C(O)--, --C(S)--, --S(O)-- or
--S(O).sub.2--; and V is absent or is --O--, --S--, --N(H)-- or
--N(Me)--, provided that V is absent when U is absent; R.sup.26 is
H, OH or R'; R' is independently selected from: H, alkyl alkenyl,
alkynyl, aryl, heteroaryl, aralkyl and heteroaralkyl; R* is
independently selected from: H and lower alkyl; q is 0 or 1; E is
an aldehyde, hydroxymethyl, carboxyl, or a protected form thereof;
P is H or a protecting group; and G is absent or represents P, or a
pharmaceutically acceptable salt thereof.
6. A compound of claim 5 having one or more of the stereochemical
configurations represented by the corresponding formula of the
group: ##STR136## ##STR137##
7. The compound of any of claims 5 or 6 wherein: R* is
independently selected from: H, methyl and ethyl; E is an aldehyde,
hydroxymethyl, carboxyl, CHO, CH.sub.2OP, CH(OP).sub.2 or
CO.sub.2P; and P is independently H or a protecting group selected
from: allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine,
phenacyl, t-butyl-diphenylsilyl, and protecting groups wherein two
occurrences of P, taken together with the atoms through which they
are connected, form a ring having 5-7 members.
8. The compound or salt of claim 7 where: R.sup.20 is H, OH,
--O.sub.2C-lower alkyl or --O.sub.2C-alkenyl; R.sup.26 is H or
CH.sub.3; R' is independently selected from: H and methyl; q is 1;
E is OPMB, TBSO--CH.sub.2-- or --C(O)H; and/or P is H, benzyl, OPMB
or TBSO.
9. The compound or salt of claim 8 where: R.sup.20 is H, OH,
--O.sub.2C--CH.sub.3, --O.sub.2C--CH.sub.2--CH.sub.2--CH.sub.3 or
--O.sub.2C--CH.dbd.CH--CH.dbd.CH--CH.sub.2--CH.sub.2--CH.sub.3.
10. The compound of salt of claim 7 where: R.sup.7 is absent;
R.sup.26 is H or C.sub.1-C.sub.6 alky; and q is zero in Formula 15
F.
11. The compound or salt of claim 7 where R.sup.26 is H.
12. A compound having the structure represented by a formula of the
group: ##STR138## ##STR139## wherein: R.sup.3 is H, OH or a
protecting group; R.sup.6 is H, H or .dbd.O; R.sup.7 is absent or
represents from 1 to 4 substituents on the ring to which it is
attached, independently selected from: lower alkyl, hydroxyl,
amino, alkoxyl, alkylamino, .dbd.O, acylamino and acyloxy; R.sup.8
is H, OH, .dbd.O, R', --(CH.sub.2).sub.nO(O)CR' or
(CH.sub.2).sub.nCO.sub.2-haloalkyl, provided that R.sup.6 and
R.sup.8 are not both .dbd.O; R.sup.9 is H, OH or is absent;
R.sup.12 and R.sup.12a are independently H, OH, lower alkyl, lower
alkoxyl, or lower acyloxy, or R.sup.12 and R.sup.12a taken together
represent .dbd.O; R.sup.20 is H, OH, or -T-U--V--R' where: T is
--O--, --S--, --N(H)-- or --N(Me)--; U is absent or is --C(O)--,
--C(S)--, --S(O)-- or --S(O).sub.2--; and V is absent or is --O--,
--S--, --N(H)-- or --N(Me)--, provided that V is absent when U is
absent; R.sup.21 is .dbd.CR.sup.aR.sup.b or R.sup.21 represents
independent moieties R.sup.c and R.sup.d where: R.sup.a and R.sup.b
are independently H, CO.sub.2R', CONR.sup.cR.sup.d or R'; R.sup.c
and R.sup.d are independently H, alkyl, alkenyl, alkynyl or
(CH.sub.2).sub.pCO.sub.2R'; R.sup.26 is H, OH or R'; R' is
independently selected from: H, alkyl, alkenyl, alkynyl, aryl,
heteroaryl, aralkyl and heteroaralkyl; L is a straight or branched
linear, cyclic or polycyclic moiety, containing a continuous chain
of preferably from 6 to 14 chain atoms, which substantially
maintains the relative distance d of about 2.5 to 5.0 angstroms
between the C1 and C17 atoms and the directionality, represented by
the bold arrows, of the C1C2 and C16C17 bonds of
naturally-occurring bryostatin; X is --CH.sub.2--, --O--, --S-- or
--N(R.sup.e)-- where R' is COH, CO.sub.2R' or SO.sub.2R', Y is
CH.sub.2, --O-- or --N(H)--; Z is --O-- or --N(H)--; n is 0, 1, 2,
3, 4 or 5; and p is 1, 2 or 3, or a pharmaceutically acceptable
salt thereof, excluding the compounds of Formula 1998a where
R.sup.3 is H or OH and where R.sup.20 is --O--C(O)--CH.sub.3 or
--O--C(O)--(CH.sub.2).sub.6--CH.sub.3, and the compounds of Formula
1998b where R.sup.8 is H or t-Bu: ##STR140##
13. A compound of claim 12 having one or more of the stereochemical
configurations represented by the corresponding formula of the
group: ##STR141## ##STR142##
14. A compound of claim 12 having the stereochemical configurations
represented by the corresponding formula of the group: ##STR143##
##STR144##
15. The compound or salt of any of claims 12 to 14 where: R.sup.7
is absent; R.sup.8 is H, alkyl, aralkyl or --O.sub.2C-lower alkyl;
R.sup.20 is H, OH, --O.sub.2C-lower alkyl or --O.sub.2C-alkenyl;
R.sup.21 is .dbd.C--CO.sub.2-lower alkyl; R.sup.26 is H or
C.sub.1-C.sub.6 alky; and X is --CH.sub.2-- or --O--.
16. The compound or salt of claim 15 where: R.sup.3 is OH; R.sup.8
is H, t-butyl, --O.sub.2C--CH.sub.3, --O.sub.2C--C(CH.sub.3).sub.3
or --O.sub.2C--CH.sub.2--CH.sub.2--CH.sub.3; and R.sup.20 is H, OH,
--O.sub.2C--CH.sub.3, --O.sub.2C--CH.sub.2--CH.sub.2--CH.sub.3 or
--O.sub.2C--CH.dbd.CH--CH.dbd.CH--CH.sub.2--CH.sub.2--CH.sub.3.
17. The compound or salt of claim 16 where R.sup.26 is H.
18. A method for preparing a bryostatin analog, comprising a step
selected from the group: ##STR145## ##STR146## wherein: R.sup.7 is
absent or represents from 1 to 4 substituents on the ring to which
it is attached, independently selected from: lower alkyl, hydroxyl,
amino, alkoxyl, alkylamino, .dbd.O, acylamino and acyloxy; R.sup.20
is H, OH, or -T-U--V--R' where: T is --O--, --S--, --N(H)-- or
--N(Me)--; U is absent or is --C(O)--, --C(S)--, --S(O)-- or
--S(O).sub.2--; and V is absent or is --O--, --S--, --N(H)-- or
--N(Me)--, provided that V is absent when U is absent; R.sup.26 is
H, OH or R'; R' is independently selected from: H, alkyl, alkenyl,
alkynyl, aryl, heteroaryl, aralkyl and heteroaralkyl; R* is
independently selected from: H and lower alkyl; q is 0 or 1; E is
an aldehyde, hydroxymethyl, carboxyl, or a protected form thereof;
and P is H or a protecting group.
19. The method of claim 18 comprising a stereospecific synthesis
wherein the starting material for Step 13a, 14a, 15c, 15d or 16d
has one or more of the stereochemical configurations represented by
formulae 12F, 12F, 15C, 15D or 16D, respectively: ##STR147##
##STR148##
20. The method of any of claims 18 or 19 wherein: Step 13a takes
place under reaction conditions including the presence of an acid;
Step 14a takes place under reaction conditions including the
presence of an acid and an alcohol of the formula R*--OH, where R*
is lower alkyl; Step 15c takes place under reaction conditions
including the presence of an acid; Step 15d takes place under
reaction conditions including the presence of an alkyl glutarate
ester; and/or Step 16d takes place under reaction conditions
including the presence of a dienolate of an ester of
acetoacetate.
21. The method of claim 20 wherein: R.sup.7 is absent; R.sup.20 is
R.sup.20 is H, OH, --O.sub.2C-lower alkyl or --O.sub.2C-alkenyl;
R.sup.26 is H or OH; R' is independently selected from: H and
methyl; R* is independently selected from: H, methyl and ethyl; q
is 1; E is OPMB, TBSO--CH.sub.2-- or --C(O)H; and/or P is H,
benzyl, OPMB or TBSO.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of co-pending
provisional application U.S. Ser. No. 60/357,177, filed Feb. 15,
2002, incorporated herein by reference. This application is also a
continuation-in-part of co-pending U.S. application Ser. No.
09/728,929, filed Nov. 30, 2000, which claims the benefit of
provisional application U.S. Ser. No. 60/168,181, filed Nov. 30,
1999, both incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention concerns biologically active compounds
related to the bryostatin family of compounds, and to methods of
preparing and utilizing the same.
[0005] 2. Introduction
[0006] Cancer is a major cause of death in the developed countries,
with more than 500,000 human fatalities occurring annually in the
United States. Cancers are generally the result of the
transformation of normal cells into modified cells that proliferate
excessively, leading to the formation of abnormal tissues or cell
populations. In many cancers, cell proliferation is accompanied by
dissemination (metastasis) of malignant cells to other parts of the
body, which spawn new cancerous growths. Cancers can significantly
impair normal physiological processes, ultimately leading to
patient mortality. Cancers have been observed for many different
tissue and cell types, with cancers of the lung, breast, and
colorectal system accounting for about half of all cases.
[0007] Currently, about one-third of cancer patients can be cured
by surgical or radiation techniques. However, these approaches are
most effective with cancerous lesions that have not yet
metastasized to other regions of the body. Chemotherapeutic
techniques currently cure another 17% of cancer patients. Combined
chemotherapeutic and non-chemotherapeutic protocols can further
enhance prospects for full recovery. Even for incurable cancer
conditions, therapeutic treatments can be useful to achieve
remission or at least extend patient longevity.
[0008] Numerous anticancer compounds have been developed over the
past several decades (e.g., Katzung, 1998; Wilson et al., 1991;
Hardman et al., 1996). While these compounds comprise many
different classes that act by a variety of mechanisms, one general
approach has been to block the proliferation of cancerous cells by
interfering with cell division. For example, anthracyclines, such
as doxorubicin and daunorubicin, have been found to intercalate
DNA, blocking DNA and RNA synthesis and causing strand scission by
interacting with topoisomerase II. The taxanes, such as Taxol.TM.
and Taxotere.TM., disrupt mitosis by promoting tubulin
polymerization in microtubule assembly. Cis-platin forms
interstrand crosslinks in DNA and is effective to kill cells in all
stages of the cell cycle. As another example, cyclophosphamide and
related alkylating agents contain di-(2-chloroethyl)-amino groups
that bind covalently to cellular components such as DNA.
[0009] The bryostatins (Formula A) are a family of naturally
occurring macrocyclic compounds originally isolated from marine
bryozoa. Currently, there are about 20 known natural bryostatins
which share three six-membered rings designated A, B and C, and
which differ mainly in the nature of their substituents at C7
(OR.sup.A) and C20 (R.sup.B) (Pettit, 1996). ##STR1## The
bryostatins exhibit potent activity against a broad range of human
cancer cell lines and provide significant in vivo life extensions
in murine xenograft tumor models (Pettit et al., 1982; Hornung et
al., 1992; Schuchter et al., 1991; Mohammad et al., 1998). Doses
that are effective in vivo are extremely low, with activities
demonstrated for concentrations as low as 1 .mu.g/kg (Schuchter et
al., 1991). Among additional therapeutic responses, the bryostatins
have been found to promote the normal growth of bone marrow
progenitor cells (Scheid, 1994; Kraft, 1996), provide cellular
protection against normally lethal doses of ionizing radiation
(Szallasi, 1996), and stimulate immune system responses that result
in the production of T cells, tumor necrosis factors, interleukins
and interferons (Kraft, 1996; Lind, 1993).
[0010] Bryostatins are also effective in inducing transformation of
chronic lymphocytic leukemia cells to a hairy cell type (Alkatib,
1993), increasing the expression of p53 while decreasing the
expression of bcl-2 in inducing apoptosis in cancer cells (Maki,
1995; Mohammad, 1995) or at least pre-disposing a cell towards
apoptosis, and reversing multidrug resistance (MDR) (Spitaler,
1998).
[0011] At the molecular level, bryostatins have been shown to
competitively inhibit the binding of plant-derived phorbol esters
and endogenous diacyl glycerols to protein kinase C (PKC) at
nanomolar to picomolar drug concentrations (DeVries, 1998), and to
stimulate comparable kinase activity (Kraft, 1986; Berkow, 1985;
Ramsdell, 1986). Unlike the phorbol esters, however, the
bryostatins do not act as tumor promoters. Thus, the bryostatins
appear to operate through a mode of action different from, and
complementary to, the modes of action of established anticancer
agents; human clinical trials are presently evaluating bryostatin
combination therapy with cisplatin or taxol.
[0012] Various studies have demonstrated good affinity for
bryostatins in which R.sup.A is hydroxyl, acetyl, pivaloyl, or
n-butanoate, and R.sup.B is H, acetyl, n-butanoate, or
2,4-unsaturated octanoate, as measured by PKC binding assay (Wender
et al., 1988). The double bond between C13 and C30 can be
hydrogenated or epoxidized without significant loss of binding
affinity. Hydrogenation of the C21-C34 alkene or acetylation of the
C26 hydroxyl, on the other hand, can significantly reduce binding
affinity. Inversion of the stereoconfiguration at C26 leads to
modest loss of activity (approx. 30-fold) and the suggestion that
the methyl group may limit rotation of bonds proximate to the
methyl group and contribute to the apparent high binding affinity
observed for the bryostatins. Elimination of the hydroxyl at C19
(with concomitant omission of the C20 R.sup.B group) causes an
approximately 100-fold to 200-fold decrease in binding. Likewise,
impairing the accessibility of the C26 hydroxymethyl moiety by
replacement of the C26 hydroxyl, or by replacing the methyl or
hydrogen substituents of C26 with a tert-butyl or similar bulky
substituent, has been proposed for diminishing toxicity (Blumberg
et al., 1997).
[0013] Although the bryostatins have been known for some time,
their low natural abundance, difficulties in isolation and severely
limited availability through total synthesis have impeded efforts
to elucidate their mode of action and to advance their clinical
development. Recently, synthetic analogues of bryostatin were
reported wherein the C4-C14 spacer domain was replaced with
simplified spacer segments using a highly efficient
esterification-macrotransacetalization (Wender et al., 1998a,
1998b). The reported analogues retained orientation of the C1-,
C19-, C26-oxygen recognition domain as determined by NMR
spectroscopic comparison with bryostatin and varying degrees of
PKC-binding affinity. The one analogue tested for in vitro
inhibition in human tumor cell lines was reported to possess
significant activity. It has remained, however, desired to provide
new, simplified, and more readily accessible synthetic agents based
on the bryostatin structure to further elucidate the molecular
basis of bryostatin's activity and develop improved and more
readily available clinical candidates, especially for anticancer
applications.
SUMMARY OF THE INVENTION
[0014] One aspect of the present invention concerns simplified
bryostatin analogues, i.e., the compounds represented by Formula I:
##STR2## wherein: [0015] R.sup.20 is H, OH, or -T-U--V--R' where:
[0016] T is selected from --O--, --S--, --N(H)-- or --N(Me)--;
[0017] U is absent or is selected from --C(O)--, --C(S)--, --S(O)--
or --S(O).sub.2--; and [0018] V is absent or is selected from
--O--, --S--, --N(H)-- or --N(Me)--, provided that V is absent when
U is absent; [0019] R.sup.21 is .dbd.CR.sup.aR.sup.b or R.sup.21
represents independent moieties R.sup.c and R.sup.d where: [0020]
R.sup.a and R.sup.b are independently H, CO.sub.2R',
CONR.sup.cR.sup.d or R'; [0021] R.sup.c and R.sup.d are
independently H, alkyl, alkenyl or alkynyl, or
(CH.sub.2).sub.nCO.sub.2R' where n is 1, 2 or 3; [0022] R.sup.26 is
H, OH or R'; [0023] R' (each instance) being independently selected
from the group: H, alkyl, alkenyl or alkynyl, or aryl, heteroaryl,
aralkyl or heteroaralkyl; [0024] L is a straight or branched
linear, cyclic or polycyclic moiety, containing a continuous chain
of preferably from 6 to 14 chain atoms, which substantially
maintains the relative distance between the C1 and C17 atoms and
the directionality of the C1C2 and C16C17 bonds of
naturally-occurring bryostatin; and [0025] Z is --O-- or --N(H)--;
and the pharmaceutically acceptable salt thereof.
[0026] In a preferred aspect of the recognition domain in this
embodiment R.sup.26 is H or methyl, particularly when R.sup.21 is
.dbd.C(H)CO.sub.2R' and/or where R.sup.20 is --O.sub.2CR'.
Especially preferred are the compounds where R.sup.26 is H. A
preferred upper limit on carbon atoms in any of R.sup.d, R.sup.e
and R' is about 20, more preferably about 10 (except as otherwise
specifically noted, for example, with reference to the embodiment
of the invention where a preferred R.sup.20 substituent has about 9
to 20 carbon atoms). In certain embodiments, R' is a straight-chain
alkyl, alkenyl (having from 1 to 6, preferably 1 to 4 double bonds,
preferably trans double bonds) or alkynyl group. In a preferred
aspect of the spacer domain of this embodiment, L contains a
terminal carbon atom that, together with the carbon atom
corresponding to C17 in the native bryostatin structure, forms a
trans olefin. It is further preferred that L contain a hydroxyl on
the carbon atom corresponding to C3 in the native bryostatin
structure.
[0027] Another aspect of the invention concerns the simplified
bryostatin analogues represented by Formulae II-V: ##STR3##
wherein: [0028] R.sup.3 is H, OH or a protecting group; [0029]
R.sup.6 is H, H or .dbd.O; [0030] R.sup.8 is selected from the
group: H, OH, .dbd.O, R', --(CH.sub.2).sub.nO(O)CR' or
(CH.sub.2).sub.nCO.sub.2-haloalkyl where n is 0, 1, 2, 3, 4 or 5,
[0031] provided that R.sup.6 and R.sup.8 are not both .dbd.O;
[0032] R.sup.9 is H, OH or is absent; [0033] R.sup.20, R.sup.21,
R.sup.26, R' and Z are as defined above with respect to Formula I;
[0034] p is 1, 2 or 3; and [0035] X is --CH.sub.2--, --O--, --S--
or --N(R.sup.e)-- where R' is COH, CO.sub.2R' or SO.sub.2R', X
preferably being --O--, and the pharmaceutically acceptable salts
thereof.
[0036] Still another aspect of the invention concerns the
simplified bryostatin analogues represented by Formulae II-A to
V-A: ##STR4## wherein: [0037] R.sup.3, R.sup.6, R.sup.9, R.sup.20,
R.sup.21, R.sup.26, R', X, Z and p are as defined above with
respect to Formulae II to V; [0038] R.sup.7 is absent or represents
from 1 to 4 substituents on the ring to which it is attached,
selected from lower alkyl, hydroxyl, amino, alkoxyl, alkylamino,
.dbd.O, acylamino, or acyloxy; [0039] R.sup.8 is as defined above
including substituted or unsubstituted alkyl, alkenyl, alkynyl,
aryl, heteroaryl, aralkyl, or heteroaralkyl, e.g., substituted with
an alkoxy, acyloxy, acylamino, or alkylamino substituent, or, in
Formula III-A when R.sup.6 represents H, H, then R.sup.8 and
R.sup.9 taken together can represent .dbd.O; [0040] R.sup.12 and
R.sup.12a are independently for each occurrence, H, OH, lower
alkyl, lower alkoxyl, or lower acyloxy, or R.sup.12 and R.sup.12a
taken together represent .dbd.O; and [0041] Y is CH.sub.2, --O-- or
--N(H)--, and the pharmaceutically acceptable salts thereof. The
compounds of Formulae II-A to V-A preferably have one or more of
the stereochemical configurations respectively represented in
Formulae II to V.
[0042] In a preferred aspect, the invention relates to the C26
des-methyl analogue of Formula IIa ##STR5## and to pharmaceutical
compositions and methods of treatment therewith.
[0043] Still another aspect of the invention relates to the C26
des-methyl homologues of the native bryostatins, as illustrated in
Formula VI: ##STR6## where R.sup.A and R.sup.B correspond to the
naturally occurring bryostatin substituents, such as C26 des-methyl
Bryostatin 1, the compound of Formula VIa: ##STR7## and to
pharmaceutical compositions and methods of treatment therewith.
[0044] Excluded from the scope of compositions of matter and
methods of treatment (as opposed to the novel synthetic processes)
in the present invention are the analogues of Formula 1998a where
R.sup.3 is H or OH and where R.sup.20 is --O--C(O)--CH.sub.3 or
--O--C(O)--(CH.sub.2).sub.6--CH.sub.3, and those of Formula 1998b
where R.sup.8 is H or t-Bu: ##STR8## Also excluded from the scope
of certain embodiments of the present invention is the subject
matter disclosed in co-pending U.S. application Ser. No.
09/728,929, filed Nov. 30, 2000 (and companion application
PCT/US00/32896 published as WO 01/40214).
[0045] In another aspect, the invention relates to a pharmaceutical
composition containing a therapeutically effective amount of a
compound of Formula I or a pharmaceutically acceptable salt thereof
admixed with at least one pharmaceutically acceptable
excipient.
[0046] In still another aspect, the invention relates to a method
of treating hyperproliferative cellular disorders, particularly
cancer in a mammal by administering to a mammal in need of such
treatment a therapeutically effective amount of a compound of
Formula I or a pharmaceutically acceptable salt thereof, either
alone or in combination with a second agent, preferably a second
anti-cancer agent that acts by a distinct mechanism vis-a-vis the
mechanism of the compound of Formula 1.
[0047] In yet another aspect, the invention relates to methods of
treatment for a mammal having an immune-related disease or
receiving immunosuppressive therapy, by administering of a
therapeutically effective amount of a compound of Formula I or a
pharmaceutically acceptable salt thereof.
[0048] In another aspect of the invention, there is provided a
method for the synthesis of bryostatin analogues, including the
steps of esterification and macrotrasacetylization of a protected
recognition domain with a protected linker synthon, followed by
deprotection. Particularly preferred is reduction of a C26 OBn
protected precursor to give the corresponding C26 des-methyl
bryostatin analogue. A related aspect of the invention entails the
novel products made by the foregoing process. Intermediates and
steps for preparing them or converting them to bryostatin analogues
are also included in the invention.
[0049] The invention also includes pharmaceutical compositions
containing one or more compounds in accordance with the
invention.
[0050] In another aspect, the invention includes a method of
inhibiting growth, or proliferation, of a cancer cell. In the
method, a cancer cell is contacted with a bryostatin analogue
compound in accordance with the invention in an amount effective to
inhibit growth or proliferation of the cell. In a broader aspect,
the invention includes a method of treating cancer in a mammalian
subject, especially humans. In the method, a bryostatin analogue
compound in accordance with the present invention is administered
to the subject in an amount effective to inhibit growth of the
cancer in the patient.
[0051] These and other objects and features of the invention will
be better understood in light of the following detailed
description.
DETAILED DESCRIPTION
Definitions
[0052] As used in the present specification, the following words
and phrases are generally intended to have the meanings as set
forth below, except to the extent that the context in which they
are used indicates otherwise.
[0053] As used herein, the terms "alkyl", "alkenyl" and "alkynyl,"
refer to saturated and unsaturated monovalent moieties in
accordance with their standard meanings, including straight-chain,
branched-chain and cyclic moieties, optionally containing one or
more intervening heteroatoms, such as oxygen, sulfur, and nitrogen
in the chain or ring, respectively. Exemplary alkyl groups include
methyl, ethyl, isopropyl, cyclopropyl, 2-butyl, cyclopentyl, and
the like. Exemplary alkenyl groups include 2-pentenyl,
2,4-pentadienyl, 2-octenyl, 2,4,6-octatrienyl,
CH.sub.3--CH.sub.2--CH.sub.2--CH.dbd.CH--CH.dbd.CH--,
cyclopentadienyl, and the like. Exemplary alkynyl groups include
CH.sub.3C.ident.CCH.sub.2--, 4-pentyn-1-yl, and the like. Exemplary
cyclic moieties include cyclopentyl, cyclohexyl, furanyl, pyranyl,
tetrahydrofuranyl, 1,3-dioxanyl, 1,4-dioxanyl, pyrrolidyl,
piperidyl, morpholino, and reduced forms of furanyl, imidazyl,
pyranyl, pyridyl, and the like.
[0054] "Lower alkyl", "lower alkenyl", and "lower alkynyl" refer to
alkyl, alkenyl, and alkynyl groups containing 1 to 4 carbon
atoms.
[0055] The term "aryl" denotes an aromatic ring or fused ring
structure of carbon atoms with no heteroatoms in the ring(s).
Examples are phenyl, naphthyl, anthracyl, and phenanthryl.
Preferred examples are phenyl and naphthyl.
[0056] The term "heteroaryl" is used herein to denote an aromatic
ring or fused ring structure of carbon atoms with one or more
non-carbon atoms, such as oxygen, nitrogen, and sulfur, in the ring
or in one or more of the rings in fused ring structures. Examples
are furanyl, pyranyl, thienyl, imidazyl, pyrrolyl, pyridyl,
pyrazolyl, pyrazinyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl,
quinoxalyl, and quinazolinyl. Preferred examples are furanyl,
imidazyl, pyranyl, pyrrolyl, and pyridyl.
[0057] "Aralkyl" and "heteroaralkyl" refer to aryl and heteroaryl
moieties, respectively, that are linked to a main structure by an
intervening alkyl group, e.g., containing one or more methylene
groups.
[0058] "Alkoxy", "alkenoxy", and "alkynoxy" refer to an alkyl,
alkenyl, or alkynyl moiety, respectively, that is linked to a main
structure by an intervening oxygen atom.
[0059] It will be appreciated that the alkyl, alkenyl, alkynyl,
aryl, heteroaryl, aralkyl, and heteroaralkyl moieties utilized
herein can be unsubstituted or substituted with one or more of the
same or different substituents, which are typically selected from
--X, --R', .dbd.O, --OR', --SR', .dbd.S, --NR'R', --NR'R'R'.sup.+,
.dbd.NR', --CX.sub.3, --CN, --OCN, --SCN, --NCO, --NCS, --NO, --NO,
.dbd.N.sub.2, --N.sub.3, --S(O).sub.2O.sup.-, --S(O).sub.2OH,
--S(O).sub.2R', --C(O)R', --C(O)X, --C(S)R', --C(S)X, --C(O)OR',
--C(O)O--, --C(S)OR', --C(O)SR', --C(S)SR', --C(O)NR'R',
--C(S)NR'R' and --C(NR)NR'R', where each X is independently a
halogen (F, Cl, Br, or I, preferably F or Cl) and each R' is
independently hydrogen, alkyl, alkenyl, or alkynyl. In one
embodiment, R' is lower alkyl, lower alkenyl, or lower alkynyl.
NR'R' also includes moieties wherein the two R' groups form a ring
with the nitrogen atom.
[0060] While practical size limits for the various substituent
groups will be apparent to those skilled in the art, generally
preferred are the alkyl, alkenyl, alkynyl, aryl, heteroaryl,
aralkyl, and heteroaralkyl moieties containing up to about 40
carbon atoms, more preferably up to about 20 carbon atoms and most
preferably up to about 10 carbon atoms (except as otherwise
specifically noted, for example, with reference to the embodiment
of the invention where a preferred R.sup.20 substituent has about 7
to 20 carbon atoms).
[0061] As to any of the above groups that contain one or more
substituents, it is understood, of course, that such groups do not
contain any substitution or substitution patterns which are
sterically impractical and/or synthetically non-feasible. In
addition, the compounds of this invention include all
stereochemical isomers and mixtures thereof arising from the
substitution of these compounds.
[0062] Except as otherwise specifically provided or clear from the
context, the term "compounds" of the invention should be construed
as including the "pharmaceutically acceptable salts" thereof (which
expression has been eliminated in certain instances for the sake of
brevity).
[0063] The term "pharmaceutically acceptable salt" refers to salts
which retain the biological effectiveness and properties of the
compounds of this invention and which are not biologically or
otherwise undesirable. In some cases, the compounds of this
invention are capable of forming acid and/or base salts, derived
from a variety of organic and inorganic counter ions well known in
the art and include, by way of example only, sodium, potassium,
calcium, magnesium, ammonium, tetraalkylammonium, and the like; and
when the molecule contains a basic functionality, salts of organic
or inorganic acids, such as hydrochloride, hydrobromide, tartrate,
mesylate, acetate, maleate, oxalate and the like.
[0064] "Relatively lipophilic," as the term is used herein,
describes a molecule, moiety, or region which is uncharged at
neutral pH, and, taken alone, is only partially soluble in water.
Relatively lipophilic moieties preferably have no more than one OH
or NH bond for every five carbon atoms, even more preferably for
every eight carbon atoms. Relatively lipophilic means that the
molecule, moiety or region facilitates therapeutic use, helping to
maintain a balance between lipophilicity (e.g., to permit cellular
uptake) and hydrophilicity (e.g., to permit aqueous
formulation).
[0065] "Mammal" is intended to have its conventional meaning.
Examples include humans, mice, rats, guinea pigs, horses, dogs,
cats, sheep, cows, etc.
[0066] The term "treatment" or "treating" means any treatment of a
disease in a mammal, including: [0067] preventing the disease, that
is, causing the clinical symptoms of the disease not to develop;
[0068] inhibiting the disease, that is, arresting the development
of clinical symptoms; and/or [0069] relieving the disease, that is,
causing the regression of clinical symptoms.
[0070] The term "effective amount" means a dosage sufficient to
provide treatment for the disease state being treated. This will
vary depending on the patient, the disease and the treatment being
effected.
Compounds
[0071] The present invention provides new analogues of bryostatin
that can be synthesized conveniently in high yields and which have
useful biological activities. The compounds of the invention can be
broadly described as having two main regions that are referred to
herein as a "recognition domain" (or pharmacophoric region) and a
relatively lipophilic "spacer domain" (or linker region). The
recognition domain contains structural features that are analogous
to those spanning C17 through C26 to C1, including the C ring
formed in part by atoms C19 through C23, and the lactone linkage
between C1 and C25 of the native bryostatin macrocycle. The spacer
domain, on the other hand, joins the atoms corresponding to C1
through C17 of the native bryostatin macrocycle to substantially
maintain the relative distance between the C1 and C17 atoms and the
directionality of the C1C2 and C16C17 bonds, as illustrated by the
arrows and distance "d" in Formula Ia (in which the substituent
groups are as defined with reference to Formula I). ##STR9## In
addition to its function of maintaining the recognition domain in
an active conformation, the spacer domain (shown as "L" in Formula
1a and sometimes also referred to as a linker region) provides a
moiety that can be readily derivatized according to known synthetic
techniques to generate analogues having improved in vivo stability
and pharmacological properties (e.g., by modulating side effect
profiles) while retaining biological activity.
[0072] It has been found in the present invention that the linker
region of the bryostatin family can be varied significantly without
eliminating activity. Thus, a wide variety of linkers can be used
while retaining significant anticancer and PKC-binding activities.
Preferably, the compounds of the present invention include a linker
moiety L, which is a linear, cyclic, or polycyclic linker moiety
containing a continuous chain of from 6 to 14 chain atoms, one
embodiment of which defines the shortest path from C25 via C1 to
C17. Distance "d" should be about 2.5 to 5.0 angstroms, preferably
about 3.5 to 4.5 angstroms and most preferably about 4.0 angstroms,
such as about 3.92 angstroms (as experimentally determined, for
example, by NMR spectroscopy). Thus, L may consist solely of a
linear chain of atoms that links C17 via C1 to C25, or
alternatively, may contain one or more ring structures which help
link C17 via C1 to C25. Preferably, the linker region includes a
lactone group (--C(.dbd.O)O--), or a lactam group
(--C(.dbd.O)NH--), which is linked to C25 of the recognition
region, by analogy to the C1 lactone moiety that is present in the
naturally occurring bryostatins. In addition, it is preferred that
the linker include a hydroxyl group analogous to the C3 hydroxyl
found in naturally occurring bryostatins, to permit formation of an
intramolecular hydrogen bond between the C3 hydroxyl of the linker
and the C.sub.1-9 hydroxyl group of the recognition region (and
optionally with the oxygen of the native B ring). In one preferred
embodiment, the linker terminates with
--CH(OH)CH.sub.2C(.dbd.O)O--, for joining to C25 of the recognition
region via an ester (or when cyclized a lactone) linkage.
[0073] In one embodiment of the invention where R.sup.26 is H, the
compounds of the invention differ from known bryostatins and
bryostatin analogues in that the present compounds contain a
primary alcohol moiety at C26, i.e., the present analogues lack a
methyl group corresponding to the C27 methyl that is ordinarily
present in naturally occurring bryostatins. Surprisingly, while the
C27 methyl moiety was previously believed to limit rotation of the
C26 alcohol and contribute to PKC binding affinity, it has been
found that this structural modification can significantly increase
PKC binding and also increases efficacy against cancer cells. Other
modifications of R.sup.26 are provided to further modulate these
characteristics, as are the C26 des-methyl homologues of the native
bryostatins.
[0074] In another aspect, the present invention provides
bryostatins and bryostatin analogues in which R.sup.20 is longer
(e.g., having 9 to 20 or more carbon atoms) than the corresponding
substituents at C20 in the native bryostatins (e.g., Bryostatin 3
having an 8-carbon atom moiety).
[0075] Certain preferred spacer domains employed in the compounds
of the invention are illustrated in Formulae II through V:
##STR10## wherein: [0076] R.sup.3 is H, OH or a protecting group;
[0077] R.sup.6 is H, H or .dbd.O; [0078] R.sup.8 is selected from
the group: H, OH, .dbd.O, R', --(CH.sub.2).sub.n(O)CR' or
(CH.sub.2).sub.nCO.sub.2-haloalkyl where n is 0, 1, 2, 3, 4 or 5,
[0079] provided that R.sup.6 and R.sup.8 are not both .dbd.O;
[0080] R.sup.9 is H, OH or is absent; [0081] R.sup.20, R.sup.21,
R.sup.26, R' and Z are as defined above with respect to Formula I;
[0082] p is 1, 2 or 3; and [0083] X is --CH.sub.2--, --O--, --S--
or --N(R.sup.e)-- where R.sup.e is COH, CO.sub.2R' or SO.sub.2R', X
preferably being --O--, and the pharmaceutically acceptable salts
thereof.
[0084] Additional embodiments of the spacer domains employed in the
compounds of the invention are illustrated in Formulae II-A to V-A:
##STR11## wherein: [0085] R.sup.3R.sup.6, R.sup.9, R.sup.20,
R.sup.21, R.sup.26, R', X, Z and p are as defined above with
respect to Formulae II to V; [0086] R.sup.7 is absent or represents
from 1 to 4 substituents on the ring to which it is attached,
selected from lower alkyl, hydroxyl, amino, alkoxyl, alkylamino,
.dbd.O, acylamino, or acyloxy; [0087] R.sup.8 is as defined above
including substituted or unsubstituted alkyl, alkenyl, alkynyl,
aryl, heteroaryl, aralkyl, or heteroaralkyl, e.g., substituted with
an alkoxy, acyloxy, acylamino, or alkylamino substituent, or, in
Formula III-A when R.sup.6 represents H, H, then R.sup.8 and
R.sup.9 taken together can represent .dbd.O; [0088] R.sup.11 and
R.sup.12a are independently for each occurrence, H, OH, lower
alkyl, lower alkoxyl, or lower acyloxy, or R.sup.12 and R.sup.12a
taken together represent .dbd.O; and [0089] Y is CH.sub.2, --O-- or
--N(H)--, and the pharmaceutically acceptable salts thereof. The
compounds of Formulae II-A to V-A preferably have one or more of
the stereochemical configurations respectively represented in
Formulae II to V.
[0090] In certain preferred embodiments, the analogues illustrated
in Formulae II-A to V-A have one or more, preferably all of the
following stereochemical dispositions: ##STR12##
[0091] Other preferred embodiments include the compounds of the
above formulae (I through V-B) where R.sup.26 is H or lower alkyl,
preferably H or Me, and most preferably H. Also preferred are those
compounds where R.sup.21 represents .dbd.CHCO.sub.2R', where R' is
preferably lower alkyl, especially Me or Et. Also preferred are
those compounds where R.sup.20 represents a carbonate, urea,
thiourea, thiocarbamate or carbamate substituent. Further preferred
are those compounds where R.sup.3 is OH. Particularly preferred are
those compounds, respectively having one or more of the
stereochemical configurations illustrated in Formulae I through
V.
[0092] Excluded from the scope of compositions of matter and
methods of treatment (as opposed to the novel synthetic processes)
in present invention are the analogues of Formula 1998a where
R.sup.3 is H or OH and where R.sup.20 is --O--C(O)--CH.sub.3 or
--O--C(O)--(CH.sub.2).sub.6--CH.sub.3, and those of Formula 1998b
where R.sup.8 is H or t-Bu: ##STR13## Also excluded from the scope
of certain embodiments of the present invention is the subject
matter disclosed in co-pending U.S. application Ser. No.
09/728,929, filed Nov. 30, 2000 (and companion application
PCT/US00/32896 published as WO 01/40214). Nomenclature
[0093] For simplicity of reference, the compounds of Formulae I-V
are named and numbered herein as corresponding to the naturally
occurring bryostatin macrocycle, described above with reference to
Formula A. For example, the C26 des-methyl homologue of native
bryostatin 1 (a compound of the present invention) has the
structure illustrated in Formula VIa: ##STR14## By way of
comparison, the analogues of the invention in which R.sup.26 is
hydrogen, such as those of Formula IIa and Formula IVa: ##STR15##
are also referred to as "C26 des-methyl", notwithstanding that the
structures corresponding to L (in Formula I) or the corresponding
spacer domain (in Formulae II-V), or even the recognition domain,
contain fewer carbon atoms than native bryostatin such that the
"C26" position would be assigned a lower number were these
analogues to be named without reference to the native structure.
Synthetic Reaction Parameters
[0094] The terms "solvent", "inert organic solvent" or "inert
solvent" mean a solvent inert under the conditions of the reaction
being described in conjunction therewith [including, for example,
benzene, toluene, acetonitrile, tetrahydrofuran ("THF"),
dimethylformamide ("DMF"), chloroform, methylene chloride (or
dichloromethane), diethyl ether, methanol, pyridine and the like].
Unless specified to the contrary, the solvents used in the
reactions of the present invention are inert organic solvents.
[0095] The terms "protecting group" or "blocking group" refer to
any group which when bound to a functional group such as one or
more hydroxyl, thiol, amino or carboxyl groups of the compounds
(including intermediates thereof) prevents reactions from occurring
at these groups and which protecting group can be removed by
conventional chemical or enzymatic steps to reestablish the
hydroxyl, thiol, amino or carboxyl group. The particular removable
blocking group employed is not critical and preferred removable
hydroxyl blocking groups include conventional substituents such as
allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine,
phenacyl, t-butyl-diphenylsilyl and any other group that can be
introduced chemically onto a hydroxyl or like functionality and
later selectively removed either by chemical or enzymatic methods
in mild conditions compatible with the nature of the product.
[0096] The term "q.s." means adding a quantity sufficient to
achieve a stated function, e.g., to bring a solution to the desired
volume (i.e., 100%).
[0097] Unless specified to the contrary, the reactions described
herein take place at atmospheric pressure within a temperature
range from about 5.degree. C. to 100.degree. C. (preferably from
10.degree. C. to 50.degree. C.; most preferably at "room" or
"ambient" temperature, e.g., 25.degree. C.). Further, unless
otherwise specified, the reaction times and conditions are intended
to be approximate, e.g., taking place at about atmospheric pressure
within a temperature range of about 5.degree. C. to about
100.degree. C. (preferably from about 10.degree. C. to about
50.degree. C.; most preferably about 25.degree. C.) over a period
of about 0.5 to about 10 hours (preferably about 1 hour).
Parameters given in the Examples are intended to be specific, not
approximate.
[0098] Isolation and purification of the compounds and
intermediates described herein can be effected, if desired, by any
suitable separation or purification procedure such as, for example,
distillation, filtration, extraction, crystallization, column
chromatography, thin-layer chromatography or thick-layer
chromatography, or a combination of these procedures. Specific
illustrations of suitable separation and isolation procedures can
be had by reference to the general description and examples.
However, other equivalent separation or isolation procedures can,
of course, also be used.
Synthesis of the Compounds of Formula I
[0099] The compounds of the invention may be produced by any
methods available in the art, including chemical and biological
(e.g., recombinant and in vitro enzyme-catalyzed) methods. In one
embodiment, the present invention provides a convergent synthesis
in which subunits primarily corresponding to the recognition and
spacer domains are separately prepared and then joined by
esterification-macrotransacetalization (Wender et al., 1998a,
1998b, 1998c). Additional syntheses of the compounds of Formulae
I-VI are described below with reference to the Reaction Schemes.
The stereochemical relationships illustrated for the various
substituents should be taken to be optional, but individually and
collectively preferred; the stereochemical relationships
corresponding to the native bryostatins are particularly
preferred.
[0100] Reaction Scheme 1 illustrates synthesis of precursors for
the recognition domain in compounds of the invention. ##STR16##
##STR17##
[0101] Reaction Scheme 2 illustrates the further synthesis of
recognition domains for C26 des-methyl compounds of the invention.
##STR18## ##STR19##
[0102] Reaction Scheme 3 illustrates synthesis of the protected
alcohol precursor to many of the C26 methyl analogues of the
invention. ##STR20## ##STR21##
[0103] Reaction Scheme 4 illustrates the synthesis of linker
synthons for preparing the compounds of Formula II. ##STR22##
##STR23##
[0104] Reaction Scheme 5A illustrates the synthesis of linker
synthons for preparing the compounds of Formula III where R.sup.9
is OH. ##STR24##
[0105] Reaction Scheme 5B illustrates the synthesis of linker
synthons for preparing the compounds of Formula III where R.sup.8
and/or R.sup.9 are H. ##STR25##
[0106] Reaction Scheme 5C illustrates the synthesis of linker
synthons for preparing the compounds of Formula III where R.sup.6
is .dbd.O with a variety of possible substituents at R.sup.8.
##STR26## ##STR27##
[0107] Reaction Scheme 6 illustrates the synthesis of linker
synthons for preparing the compounds of Formula IV. ##STR28##
[0108] Reaction Scheme 7A illustrates the synthesis of the
compounds of Formula II and II-A. ##STR29##
[0109] Reaction Scheme 7B illustrates the synthesis of the
compounds of Formula III. ##STR30##
[0110] Reaction Scheme 8 illustrates synthesis of the Compounds of
Formula IV, particularly where R.sup.26 is methyl, the C26
des-methyl analogues and compounds of Formula IV where R.sup.12 and
R.sup.12a are other than H being obtained by like synthesis.
##STR31## ##STR32##
[0111] Reaction Scheme 9 illustrates synthesis of the Compounds of
Formula V. ##STR33##
[0112] Reaction Schemes 10 and 11 illustrate the synthesis of
compounds of the invention that are further derivatized at the C20
position, as discussed in Examples 4B, 4C and 4D. ##STR34##
##STR35## ##STR36##
[0113] Starting Materials. Conveniently, compounds of the invention
can be prepared from starting materials that are commercially
available or may be readily prepared by those skilled in the art
using commonly employed synthetic methodology.
[0114] Reaction Scheme 1 illustrates a method for forming a synthon
designated herein as 111 which is useful for providing the
recognition domain in compounds of the invention, for example as
detailed in Example 1.
6-(Tert-butyldimethylsilylhydroxy)-5-dimethylhexane-2,4-dione (101,
Example 1B) is stirred with 2 equivalents of LDA (lithium
diisopropylamine) in THF (tetrahydrofuran), followed by addition of
0.9 equivalents of 3R-p-methoxybenzyl-4R-benzylhydroxypentane-1-al
(102, Example 1A) to afford diasteriomeric aldol mixture 103 after
suitable purification. To 103 is then added a catalytic amount of
p-methylphenylsulfonic acid (p-TsOH) with stirring at room
temperature followed by base quenching to produce pyranone
condensation product 104 as a mixture of .alpha. and .beta.-isomers
at C23 (104a and 104b). The .beta.-isomer (104a) is separated from
the .alpha.-isomer and is reacted with NaBH.sub.4 in the presence
of CeCl.sub.3-7H.sub.2O, followed by quenching with aqueous brine
to form an allylic alcohol (not shown) that can then be epoxidized
with m-chloroperbenzoic acid (mCPBA) in 2:1 CH.sub.2Cl.sub.2:MeOH
containing sodium bicarbonate as a buffer to yield a
C19-methoxylated C20, C21 syn-diol 105. Selective benzoylation of
the C21 equatorial alcohol with benzoyl chloride to afford C21
monobenzoate (not shown), followed by oxidation of the C20 hydroxyl
with Dess-Martin periodinane
(1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one) at room
temperature affords the corresponding 20-keto-21-benzoate product
106. Treatment of 106 with SmI.sub.2 (2 equiv) yields a ketone 107
selectively deoxygenated at C21. Next, ketone 107 is reacted with
LDA and OHCCO.sub.2CH.sub.3 in THF at -78.degree. C. to afford
aldol mixture 108. After purification, 108 is reacted with
methanesulfonylchloride in CH.sub.2Cl.sub.2 containing
triethylamine, followed by reaction with DBU
(1,8-diazabicyclo[5.4.0]undec-7-ene) in THF to effect an aldol
condensation and elimination of water, to yield an
.alpha.,.beta.-unsaturated methyl ester (enone 109) with an
E-stereoconfiguration. Treatment of enone 109 with NaBH.sub.4 in
the presence of CeCl.sub.3-7H.sub.2O produces exclusively the C20
axial alcohol 110. This product can then be esterified at C20, with
octanoic acid for example, to yield the desired synthon 111.
[0115] It will be appreciated how the foregoing procedures can be
exploited or modified to produce recognition region synthons having
different substituents. For example, compounds where R.sup.21
contains a C35 ester group having a Z-configuration are produced
during formation of intermediate 109 (Example 1C) and can be
isolated by chromatography. Similarly, other ester groups can be
introduced at C35 by replacing the OHCCO.sub.2CH.sub.3 reactant
used to form 108 above with an appropriately substituted compound
of the form OHCCO.sub.2R', in which R' is other than methyl.
[0116] In addition, as detailed below, other substituents can be
introduced in synthon 111 to generate substituent R.sup.20 at C20
by substituting any of a variety of carboxylic acids for the
octanoic acid reacted with axial alcohol 110 (as in the last step
of Example 1C), including other saturated, unsaturated, aryl, and
carboxylic acids. In synthesizing the compounds of the invention
where R.sup.20 has been varied, the substituent (e.g., a desired
C20 ester substituent, carbonate, urea, thiourea, thiocarbamate or
carbamate) can be introduced into a recognition region synthon
prior to condensing the recognition region synthon with a linker
synthon using the procedures described in Example 4 and via other
synthetic routes well known to those skilled in the art. In Example
4A, the C20 octanoate substituent in synthon 111 can be replaced
with an acetyl group by first protecting the base labile aldehyde
group using trimethyl orthoformate to form the dimethyl acetal. The
C20 octanoate ester can then be cleaved using a basic solution,
such as K.sub.2CO.sub.3 in methanol, to afford the free C20
alcohol, followed by reaction with an activated form of acetic
acid, such as acetic anhydride or acetyl chloride, to obtain the
C20 acetate product. The product can then be deprotected at the C15
aldehyde, C19 oxygen, and C25 oxygen using benzoquinone compound
DDQ (to remove the p-methoxybenzyl group and cleave the dimethyl
acetal) followed by aqueous HF (demethylation at C19) to afford the
corresponding C19 alcohol. This product can then be condensed with
an appropriately substituted linker synthon to produce a desired
bryostatin analogue, such as analogue 702.1, as detailed in Example
4A.
[0117] The protected alcohol precursor to many of the C26-desmethyl
bryostatin analogues of the invention (the compounds of Formula I
where R.sup.26 is H) can be made as illustrated in Reaction Scheme
2. Di(benzyl ether) 111 can be hydrogenated over Pearlman's
catalyst to produce the corresponding C25, C26 diol 201. Treatment
of the diol with lead tetraacetate yields the corresponding C25
aldehyde (not shown), with the release of C26 and C27. Reaction of
the aldehyde with Cp.sub.2Ti(Cl)CH.sub.2Al(CH.sub.3).sub.2 (Tebbe's
reagent) yields C25,C26 olefin 202. Alternatively, sodium periodate
can be used in place of lead tetraacetate.
[0118] Treatment of olefin 202 with HF/pyridine is effective to
remove the silyl protecting group, followed by treatment with
Dess-Martin periodinane (supra) to oxidize the C17 alcohol to an
aldehyde group, affording aldehyde 203. The C25,C26 olefin of 203
can be converted to C25,C26 diol 204 by reaction with chiral
dihydroxylating reagent (DHQD).sub.2AQN in the presence of
K.sub.3Fe(CN).sub.6, K.sub.2CO.sub.3 and K.sub.2OsO.sub.2(OH).sub.4
in t-butanol. Product 204 is obtained as a 2:1 (.beta.:.alpha.)
mixture of 25-hydroxy diastereomers. The .alpha.-diastereomer can
be removed later in the synthesis. Treatment of 204 with
triethylsilyl chloride yields protected diol 205, which can be
employed in the synthesis of the compounds of Formula V.
[0119] Addition of backbone atoms corresponding to C15 and C16 of
the bryostatin backbone to 205 can be accomplished in four steps.
First, the C17 aldehyde is allylated with allyl diethylborane. The
reaction is quenched with saturated sodium bicarbonate to yield the
desired C17 allyl adduct. The C17 hydroxyl group can then be
acylated with acetic anhydride in the presence of triethylamine and
4-dimethylaminopyridine (DMAP), to afford a diastereomeric mixture
of homoallylic C17 acetates. This product mixture can be oxidized
using N-methylmorpholine N-oxide and osmium tetraoxide, followed by
neutralization with sodium bicarbonate. After extraction, the
residue is reacted with lead tetraacetate, followed by addition of
DBU to cause elimination of the acetate group, yielding enal
206.
[0120] The C25 hydroxyl group of 206 can be unmasked in preparation
for closure of the macrocycle as follows. First, enal 206 is
treated with aqueous hydrofluoric acid to provide a crude diol
intermediate in which the C19 methoxy group is converted to a free
hydroxyl. Next, the diol product is reacted with
tert-butyldimethylsilyl chloride (TBSCl) in the presence of
imidazole to produce alcohol 207 containing a C25 hydroxyl group
and C26 OTBS group as a 2:1 (.beta.:.alpha.) mixture of C25
diastereomers. Silica gel chromatography can be used to resolve the
diastereomers, affording the .beta. diastereomer in 50-60%
yield.
[0121] The protected alcohol precursor to many of the C26-methyl
bryostatin analogues of the invention (Formula I where R.sup.26 is
methyl) can be made as illustrated in Reaction Scheme 3, via
methods analogous to the preparations for 205 and 206. Deprotection
and acylation of formula III may be accomplished, for example, by
following the procedures described in Wender et al., (1998a).
[0122] Linker synthons for the compounds of Formula II (where X is
a heteroatom) can be prepared, for example, as illustrated with
reference to Reaction Scheme 4, and later described in Examples 2A
and 2B. These compounds contain two rings that are analogous to the
A and B rings of bryostatin, but lack the naturally occurring
substituents at C7, C8, C9, and C13. The presence of a heteroatom,
such as an oxygen, sulfur or nitrogen atom (the lone electron pair
of which is stabilized) in place of C14 does not adversely affect
activity of the end product, but is required for transacetylization
in the later synthetic steps. The compounds of formulae 406 and 408
differ in that 402 provides a protecting group precursor for a
hydroxyl group attached to C3, whereas 406 does not provide for a
hydroxyl at C3.
[0123] The linker synthons for the compounds of Formula III (in
which X is a heteroatom), which contain a B-ring-like structure but
lack an A-ring, are prepared, for example, as illustrated with
reference to Reaction Schemes 5A through 5C. Examples 2C and 2D
describe methods for preparing synthons 504 and 508. In both
examples, R.sup.8 is a tert butyl group attached to C9. However,
with reference to the preparation of 505, the t-BuLi reactant can
be replaced by R'Li to generate the corresponding linker synthons
of 508 where R.sup.8 is R'. 504 additionally contains a TMS
protecting group for synthesis of the compounds where R.sup.9 is a
hydroxyl attached to C9, rather than hydrogen. Also, both synthons
contain a TBS protecting group for the compounds where R.sup.3 is a
hydroxyl group attached to C3. Example 2E describes the
corresponding method for making synthon 507, which is unsubstituted
at C9. Example 2G describes a method for preparing linker synthons
in which C5 is provided as an ester carbonyl. In addition, the
synthons in this Example contain an R.sup.6 substituent that is
preferably a saturated or unsaturated substituent containing 1 to
20 carbon atoms and optionally (1) one or more oxygen atoms and (2)
optionally one or more nitrogen atoms. In synthon 514 in Example
2G, R.sup.8 is
--C(CH.sub.3).sub.2CH.sub.2C(.dbd.O)C.sub.13H.sub.27. However,
other R.sup.6 substituents can be introduced by suitable
modification of the procedure as will be evident to one of ordinary
skill in the chemical arts.
[0124] Synthesis of a completely acyclic linker synthon 606 (where
neither an A- nor a B-ring-like structure is present) is described
with reference to Reaction Scheme 6 and in Example 2F.
[0125] As illustrated with reference to Reaction Scheme 7A, and
further described in Example 3A, an alcohol such as 207, 303 or 304
is reacted with an acid such as 406 or 408 in a two step process to
form the desired macrocyclic structure. After in situ conversion of
the acid (408) to a mixed anhydride, the alcohol (207) is added to
form ester 701. The ketal portion of 408 is then joined (in a
process referred to as macrotranacetylization) to C15 of 701 by
adding 70% HF/pyridine hydrofluoric acid to catalyze cleavage of
the menthone ketal, cleavage of the TBS ethers at C3 and C26, and
formation of a new ketal between the C15 aldehyde group and the
linker diol moiety generated by release of the menthone (where X is
oxygen), to afford desired analogue of Formula II where X is a
heteroatom and R.sup.26 is H (starting with alcohol 207) or methyl
(starting with 303 or 304), i.e., compound of formula 702. This
last reaction is also effective to set the stereocenter at C15 to a
thermodynamically preferred configuration. The analogous synthesis
of compounds of Formula III (where X is a heteroatom), first
forming the ether bond between C1 and C25, is illustrated with
reference to Reaction Scheme 7B (where formula 703 corresponds to
any of formulae 504, 507, 508 or 513) and further described in
Example 3C.
[0126] As illustrated with reference to Reaction Scheme 8, and
further described in Example 3B, the compounds of Formula IV (such
as formula 807) can be made from pharmacophoric synthon 801 and
linker synthon 606 from Example 2F.
[0127] Reaction Scheme 9 illustrates synthesis of the compounds of
Formula V, e.g., as further described in Example 3D, from synthon
111 and an activated dicarboxylic acid (succinic anhydride) to give
formula 903.
[0128] Although the bryostatin analogues produced in Examples 3B,
3C and 3D all contain a C27 methyl group, analogous C26 desmethyl
analogues can be readily synthesized using an appropriate C26
desmethyl synthon, such as C26 desmethyl synthon 207 described in
Example 1C. Compounds of the invention having a naturally occurring
bryostatin backbone (e.g., including a naturally occurring linker
region), but lacking the C27 methyl group, can also be prepared by
adapting synthetic protocols published by Kageyama et al. (1990)
and Evans et al. (1998), which are incorporated herein by
reference. In brief, the published methods are modified by
utilizing synthons in which the C27 methyl group has been omitted,
to afford the desired C26-desmethyl analogues.
[0129] As illustrated with reference to Reaction Scheme 10,
synthesis of a C20 heptanoate ester 43 is described in Example 4B,
using a similar reaction scheme to that employed in Example 4A,
except that heptenoic acid in the presence of triethylamine, DMAP,
and Yamaguchi's agent is used in place of acetic anhydride.
Yamaguchi's reagent is again employed in step f to activate the
COOH group of formula 6, followed by removal of the TBS group in
step g, hydrolysis of the menthone and transacetylization in step
h, and saturation of the double bond upon removal of the benzyl
group by hydrogenolysis in step i. Synthesis of a C20 myristate
ester analogue 48 (14 carbon atom chain) is illustrated with
reference to Reaction Scheme 11 and described in Example 4C.
Reaction Scheme II and Example 4D describes synthesis of a
bryostatin analogue containing an aryl ester group (benzoate) at
C20, by suitable adaptation of the procedure in Example 1C for
making compound 207. It will be appreciated how these procedures
can be modified to introduce other C20 esters by substituting the
starting materials necessary to produce the desired products. In
particular, C26 des-methyl analogues can be made using an
appropriate C26 des-methyl synthon, such as 207 noted above.
[0130] The lactam analogues of the invention (wherein Z attached to
C25 is NH) are obtained by converting the C.sub.2-5 hydroxyl group
(e.g., of formula 207) to an amine under Mitsonobu conditions,
after first protecting the aldehyde (and the C19 hydroxyl group in
the corresponding compounds in Reaction Schemes 10 and 11) followed
by formation of the macrocycle and de-protection under conditions
analogous to those employed for the lactone analogues, as will be
apparent to one skilled in the art. Lactam embodiments, can also be
prepared by performing an aminohydroxylation reation, such as has
been disclosed by Sharpless et al., instead of dihydroxylation with
OsO.sub.4. (employing protection/deprotection as described above).
Chiral ligands for this reaction are known, and can be used to
influence the stereochemical and/or regiochemical outcomes of the
aminohydroxylation. This strategy can be employed on substrates in
which the terminal alkene of the above scheme is further
substituted, thereby providing access to compounds wherein R.sup.26
is other than hydrogen. Such starting materials can be prepared by
cleaving the olefin to the aldehyde (e.g., by OsO.sub.4/periodate
or ozonolysis) and performing a Wittig or other olefination
reaction to obtain a desired secondary alkene.
[0131] The C26 des-methyl bryostatin homologues of the invention
can be obtained by substituting homologous des-methyl starting
materials for the starting materials employed in published
bryostatin syntheses (e.g., Masamune 1988a, 1988b, Evans et al.
1998, Kageyama et al. 1990). For example, in the total synthesis of
bryostatin 7, Serine is substituted for threonine in a Masamune's
C17-C26 southern bryostatin synthesis to yield the corresponding
C26 des-methyl sulfone. Other synthetic methodology will be
apparent to those skilled in the art given the objective of
providing such C26 des-methyl bryostatin homologues.
[0132] In certain embodiments, the fragment corresponding to the
recognition domain (or C-ring portion) of bryostatin is prepared by
a method that includes one or more of the steps illustrated with
regard to Reaction Schemes 12 to 15, wherein: [0133] R', R.sup.20
and R.sup.26 are as defined above (for example, R.sup.20 being
--O.sub.2CR' or --O.sub.2CNHR'); [0134] R* represents,
independently for each occurrence, H or a lower alkyl group such as
methyl or ethyl; [0135] q represents 0 or 1; [0136] E represents an
aldehyde, hydroxymethyl, carboxyl group, or a protected form
thereof, such as CHO, CH.sub.2OP, CH(OP).sub.2, or CO.sub.2P;
[0137] P represents H or a protecting group, including protecting
groups wherein two occurrences of P, taken together, form a ring
having 5-7 members including the atoms through which they are
connected (e.g., CH(OP).sub.2 may represent a cyclic acetal, e.g.,
with ethylene glycol or pinacol); and [0138] G is absent or
represents P.
[0139] With respect to the reactions illustrated in Reaction
Schemes 13 to 15, the group identified as R' is preferably hydrogen
or lower alkyl (such as methyl or ethyl).
[0140] In certain embodiments wherein P is a protecting group for a
hydroxyl, P represents a trialkylsilyl, dialkylarylsilyl, benzyl,
substituted benzyl, benzhydryl, substituted benzhydryl,
5-dibenzosuberyl, triphenylmethyl, substituted triarylmethyl,
naphthyldiphenylmethyl, 2- or 4-picolyl,
3-methyl-2-picolyl-N-oxide,
4-(4'-bromophenacyloxyphenyl)diphenylmethyl,
4,4',4''-tris(4,5-dichloro-phthalamidophenyl)methyl,
4,4',4''-tris(levulinoyloxyphenyl)methyl,
4,4',4''-tris(benzoyloxy-phenyl)methyl, 9-(9-phenyl)xanthenyl,
pivaloyl, adamantoyl, and 2,2,2-trichloroethylcarbonyl.
[0141] In certain embodiments wherein P is a protecting group for
an acetal, P represents lower alkyl (such as methyl, ethyl,
isopropyl, etc.) or, taken together with a second occurrence of P,
represents a lower alkylene group (such as CH.sub.2CH.sub.2,
CH.sub.2CMe.sub.2CH.sub.2, etc.).
[0142] In certain embodiments wherein P is a protecting group for a
carboxylic acid, P represents dialkylarylsilyl, benzyl, substituted
benzyl, trichloroethyl, t-butyl, trimethylsilylethyl, lower alkyl,
lower alkenyl (such as allyl), or other suitable protecting
group.
[0143] In certain embodiments, E represents a carboxylic acid
derivative that can be readily converted to an aldehyde, such as a
thioester (reduction by triethylsilane in the presence of a
transition-metal catalyst), CONMe(OMe) (reduction by a hydride
reagent), an ester (reduction by diisobutylaluminum hydride
(DIBAL-H)), etc. Other such groups and reaction conditions are well
known to those of skill in the art.
[0144] The starting materials for Reaction Schemes 13 and 14 can be
prepared, for example, as illustrated in Reaction Scheme 12.
##STR37##
[0145] In one embodiment, compound 12A can be converted to compound
12B by reaction with an acetone enolate or equivalent thereof, such
as a lithium or magnesium anion of the N,N-dimethylhydrazone of
acetone. Alternatively, the methyl ester of 12A can be reduced to
an aldehyde (e.g., by reduction with DIBAL-H, or reduction by
lithium aluminum hydride (LAH) followed by Swern oxidation),
followed by aldol addition with an acetone enolate or equivalent
and oxidation of the aldol product to the diketone. The dienolate
of diketone 12B can then be reacted with aldehyde 12C to give aldol
product 12F, which can be subsequently protected and/or reduced at
the C.sup.21 ketone selectively (e.g., with tetramethylammonium
triacetoxyborohydride, with an aldehyde in the presence of a Lewis
acid via an intramolecular hydride transfer, etc.).
[0146] Alternatively, ester 12A can be converted to ketone 12E,
e.g., by reduction to the aldehyde, reaction with methyllithium or
methyl magnesium halide, and oxidation to the ketone. Aldehyde 12C
can be extended to aldehyde 12D by aldol addition of an
acetaldehyde equivalent (e.g., reaction with an alkylsilane,
allylborane, or allyltin reagent followed by ozonolysis or
dihydroxylation/periodate cleavage, reaction with a silyl ketene
acetal of an acetate ester in the presence of a Lewis acid catalyst
such as TiCl.sub.3(OiPr), TiCl.sub.2(OiPr).sub.2, SnCl.sub.4, or
Sn(OTf).sub.2 followed by reduction to the aldehyde, reaction of an
enolate of an acetate ester followed by reduction to the aldehyde,
etc. For many of these reactions, chiral reagents or ligands are
available that may be used to favor a desired diastereomer of the
aldol product, while others may be sufficiently stereoselective
without use of a chiral reagent. An enolate of ketone 12E can then
be added to aldehyde 12D to arrive at aldol 12F by a different
route.
[0147] Compounds wherein q is 0 can be readily converted to
compounds wherein q is 1 as is described in detail above, e.g., by
converting 12E to an aldehyde and performing a Horner-Emmons
reaction with a phosphonoacetate reagent, performing a Peterson
reaction with a trimethylsilylacetate reagent, or by other
techniques known to those of skill in the art or described
above.
[0148] Compounds useful in executing this strategy also include
compounds represented by Formula 12G: ##STR38## where, as valence
and stability permit, q, E, P, G and R.sup.26 are as defined above
(with and without regard to the illustrated stereochemical
relationships).
[0149] In Reaction Scheme 13 (below) Step a can be performed by
treating the diketone compound 12F (produced as described in
Reaction Scheme 12, where G at C.sub.21 is absent) with acid,
preferably under conditions that favor the removal of the water
generated, such as distillation of the generated water (optionally
as an azeotrope), addition of a dehydrating agent (such as
molecular sieves, a carboxylic acid anhydride, or sodium or calcium
sulfate), or other suitable means. If P on the C.sub.23 hydroxyl
represents a protecting group, the cyclization can be performed
under conditions that selectively remove this protecting group, or
this protecting group can be removed prior to the cyclization.
##STR39##
[0150] Step b can be performed by a series of reactions. For
example, step b may begin with reduction of the ketone 13A with a
hydride source, such as lithium or sodium borohydride, lithium
aluminum hydride, etc. In certain embodiments, a reduction
selective for the ketone over the unsaturation is performed, such
as a Luche reduction (sodium borohydride in the presence of cerium
(III) chloride heptahydrate). Oxidation of the alkene can then be
performed with monoperoxyphthalate hexahydrate (MMPP), by
epoxidation (e.g., with mCPBA, or dimethyldioxirane), or by
dihydroxylation (e.g., with osmium tetroxide, optionally with an
asymmetric ligand as is well known in the art). The product of the
oxidation, if performed in the presence of water, will be the
hemiketal, and if performed in the presence of an alcohol, will be
the corresponding mixed ketal. These and other reactions necessary
to perform step b can be conducted in analogy with Wender et al.,
J. Am. Chem. Soc. 1998, 120, 4534-4535.
[0151] The present invention also encompasses the intermediate
compounds useful in executing this strategy, including the
compounds of Formulae 13A and 13B (with and without regard to the
illustrated stereochemical relationships).
[0152] Compounds where R.sup.20 is H can be prepared by a method
including one or more steps of the following sequence:
##STR40##
[0153] Step a can be performed starting with a compound of Formula
12F (produced as described in Reaction Scheme 12, where G at
C.sub.21 is hydrogen) by an acid-catalyzed cyclization in a
reaction mixture including an alcohol HOR*, preferably as a solvent
or cosolvent, in order to form the mixed ketal. If P on the C23
hydroxyl represents a protecting group, the cyclization can be
performed under conditions that selectively remove this protecting
group (and possibly also any protecting group at C21), or one or
both of these protecting groups can be removed prior to the
cyclization.
[0154] Step b can be performed by oxidation of the C21 alcohol
(e.g., by Swern oxidation, Dess-Martin periodinane, etc.), after
removing any protecting group at this position, followed by
reaction with a reagent such as R'O.sub.2CCH.sub.2SiMe.sub.3 or
R'O.sub.2CCH.sub.2PO.sub.3Me.sub.2. Optionally, a chiral
phosophonoacetate (e.g., a phosphonate ester of BINOL, or other
chiral diols or alcohols) or phosphonamidoacetate (e.g., a
phosphonate derivative of ephedrine or another chiral aminoalcohol)
may be employed in order to favor the desired stereochemical
outcome of the enoate installation. Subsequent steps may be
performed in analogy to well known procedures as discussed
above.
[0155] In embodiments wherein q is 0, extension to embodiments
wherein q is 1 can be readily accomplished by reaction of a
compound wherein E is an aldehyde and q is 0 with a reagent such as
Li--CH.dbd.CHOEt, ECH.sub.2P(O)(OMe).sub.2, ECH.sub.2SiMe.sub.3,
etc., wherein E represents an aldehyde or ester moiety. In certain
preferred embodiments, a compound wherein E is aldehyde and q is 0
is converted to a compound wherein E is aldehyde and q is 1 by
treatment with a preparation of a 2-alkoxyvinyllithium (such as
2-ethoxyvinyllithium) and a dialkylzinc (such as dimethylzinc),
followed by treatment with acid, such as HCl, to convert the
beta-hydroxyenol ether adduct to the unsaturated aldehyde, as is
described in greater detail below. Other suitable reagents will be
well known to those of skill in the art.
[0156] The present invention also encompasses the intermediate
compounds useful in executing this strategy, including the
compounds of Formulae 14A and 14B (with and without regard to the
illustrated stereochemical relationships).
[0157] Alternatively, the C-ring fragment analog for compounds
where R.sup.26 is H can be prepared as illustrated in Reaction
Scheme 15. ##STR41##
[0158] In this scheme, alcohol 15A can be oxidized to an aldehyde
(e.g., by Swern or TPAP/NMO oxidation), followed by addition of a
Grignard or lithium reagent derived from a 4-halo-1-butanol, such
as 4-chloro- or 4-bromo-1-butanol. Both alcohols of 15B can then be
oxidized (e.g., by Swern or TPAP/NMO oxidation), followed by
selective addition of an allyl group to the aldehyde (e.g., by
treatment with an allylstannane or allylsilane in the presence of a
Lewis acid catalyst, optionally in the presence of a chiral ligand
for the Lewis acid, such as BINOL, Ti(OPr).sub.4, and B(OMe).sub.3
together) to give ketoalcohol 15C. Cyclization and elaboration of
this piece can be performed in analogy with the scheme presented
above, for example, by cyclizing in the presence of an acid under
dehydrating conditions, followed by oxidation of the enol ether
with magnesium monoperoxyphthalate hexahydrate (MMPP), and
oxidation of the resulting alcohol (e.g., by Swern or TPAP/NMO
oxidation) to give ketone 15D. The exocyclic enoate can then be
installed by an aldol condensation with methyl glyoxylate, e.g., by
forming the lithium anion of ketone 15D with lithium
diisopropylamide (LDA) under anhydrous conditions, or in an
alcoholic solvent (such as methanol) in the presence of a base
(such as sodium, potassium, or cesium carbonate). The terminal
alkene can then be oxidized to the diol (e.g., by OsO.sub.4 or
certain hypervalent iodine reagents), optionally in the presence of
a chiral ligand, as is well known in the art. Embodiments wherein q
is 0 can be converted to embodiments wherein q is 1 as described in
detail above.
[0159] The present invention also encompasses the intermediate
compounds useful in executing this strategy, including the
compounds of Formulae 15C, 15D, 15E and 15F (with and without
regard to the illustrated stereochemical relationships).
[0160] In certain embodiments, a fragment analogous to the A and B
rings of bryostatin can be prepared for incorporation into an
analog of the present invention by a method including the steps
illustrated in Reaction Scheme 16, where the substituents R*,
R.sup.7 and P are as defined above: ##STR42##
[0161] Thus, a glutaric diester 16A can be condensed with a
dienolate of an acetoacetate ester to provide diketoester 16B,
followed by reduction of the two ketones to give diol 16C. One
ketone can be reduced by a Noyori hydrogenation in the presence of
a chiral catalyst to impart a first asymmetric center, and the
second can be reduced stereoselectively using the newly formed
stereocenter (tetramethylammonium triacetoxyborohydride for anti,
Et.sub.2BOMe and NaBH.sub.4 for syn, for example). Acid-catalyzed
lactonization followed by protection of the remaining alcohol to
give lactone 16D provides a substrate for a second reaction with a
dienolate of an acetoacetate ester to give ketoester 16E.
Acid-catalyzed reduction of the hemiketal with triethylsilane, or
dehydration of the hemiketal followed by hydrogenation, generates
the tetrahydropyran of the A-ring analog. The remaining ketone can
be reduced using a Noyori hydrogenation for a second time, or by
hydride reduction in the presence of a chelating Lewis acid to take
advantage of the tetrahydropyran stereochemistry, thereby producing
alcohol 16F. Optionally, the terminal 1,3-diol can be converted to
a ketal 16G in the presence of acid and a ketone, ketal, or enol
ether.
[0162] The present invention also encompasses the intermediate
compounds useful in executing this strategy, including the
compounds of Formulae 16E, 16F and 16G (with and without regard to
the illustrated stereochemical relationships). Compounds useful in
executing this strategy also include compounds represented by
Formulae 16H, 16I and 16J: ##STR43##
Preferred Processes and Last Steps
[0163] A C19,C26 hydroxyl-protected, C26 des-methyl bryostatin
recognition domaine precursor and an optionally protected linker
synthon are esterified, macrotransacetylated and de-protected to
give the corresponding C26 des-methyl bryostatin analogue.
[0164] A bryostatin analogue precursor having the C26 hydroxyl
substituted by a protecting group (particularly OBn) is reduced to
give the corresponding compound of Formula I.
[0165] Serine is substituted for threonine in a Masamune's C17-C26
southern bryostatin synthesis to yield the corresponding C26
des-methyl sulfone, which in turn is employed in synthesis of a C26
des-methyl bryostatin homologue.
[0166] A pyran-4-ol of Formula 404 is converted to the
corresponding pyran-2-yl-acetaldehyde of Formula 405 under reaction
conditions including the presence of isobutylvinyl ether and Hg(II)
diacetate. The reaction conditions further include carrying the
crude vinylated pyran forward without delay, contacting it with
anhydrous decane.
[0167] A diketone of Formula 12F is converted to the corresponding
dihydropyranone of Formula 13A under reaction conditions including
the presence of an acid, particularly where R.sup.26 represents H
or C.sub.1 to C.sub.6 alkyl. The reaction conditions can further
include means for removing water.
[0168] A ketone of Formula 12F is converted to a tetrahydropyran of
Formula 14A under reaction conditions including the presence of an
acid and an alcohol (R*OH), particularly where R.sup.26 represents
H or C.sub.1 to C.sub.6 alkyl. The alcohol can be present as a
solvent or cosolvent making up at least 20% of total solvent.
[0169] A ketone of Formula 15C is converted to the corresponding
dihydropyranone of Formula 15D under reaction conditions including
the presence of an acid. The reaction conditions can further
include means for removing water.
[0170] A ketone of Formula 15D is converted to the corresponding
ketoenoate of Formula 15E under reaction conditions including the
presence of an alkyl glutarate ester.
[0171] An optionally protected lactone of Formula 16E is contacted
with a dienolate of an ester of acetoacetate under conditions that
provide the corresponding tetrahydropyran of Formula 16F.
[0172] A compound of Formula I-VI is contacted with a
pharmaceutically acceptable acid to form the corresponding acid
addition salt.
[0173] A pharmaceutically acceptable acid addition salt of Formula
I-VI is contacted with a base to form the corresponding compound of
Formula I-VI.
[0174] Also preferred is a stereospecific synthesis for preparing a
bryostatin analog, having a step selected from the group: ##STR44##
particularly where the starting material for Step 13a, 14a, 15c,
15d or 16d has one or more of the stereochemical configurations
represented by formulae 12F, 12F, 15C, 15D or 16D, respectively,
and especially where:
[0175] R.sup.7 is absent;
[0176] R.sup.20 is R.sup.20 is H, OH, --O.sub.2C-lower alkyl or
--O.sub.2C-alkenyl;
[0177] R.sup.26 is H or OH;
[0178] R' is independently selected from: H and methyl;
[0179] R* is independently selected from: H and methyl;
[0180] q is 1;
[0181] E is OPMB, TBSO--CH.sub.2-- or --C(O)H; and/or
[0182] P is H, benzyl, OPMB or TBSO.
Further preferred are those processes where:
[0183] Step 13a takes place under reaction conditions including the
presence of an acid; [0184] Step 14a takes place under reaction
conditions including the presence of an acid and an alcohol of the
formula R*--OH, where R* is lower alkyl; [0185] Step 15c takes
place under reaction conditions including the presence of an acid;
[0186] Step 15d takes place under reaction conditions including the
presence of an alkyl glutarate ester; and/or [0187] Step 16d takes
place under reaction conditions including the presence of a
dienolate of an ester of acetoacetate.
Preferred Compounds
[0188] The following substituents, compounds and groups of
compounds are presently preferred. In the compounds of Formulae
I-V, especially those of Formulae II-V, it is preferred that
R.sup.26 is H. Most preferred are the compounds of Formula II where
R.sup.26 is H, and of those where X is oxygen. Of the compounds
where R.sup.26 is H, additionally preferred are those compounds
where R.sup.20 is O.sub.2CR', especially where R' is alkyl
(preferably about C.sub.7-C.sub.20 alkyl), alkenyl (preferably
about C.sub.7-C.sub.20 alkenyl such as
CH.sub.3--CH.sub.2--CH.sub.2--CH.dbd.CH--CH.dbd.CH--) or aryl
(preferably phenyl or naphthyl). Another group of preferred
compounds where R.sup.26 is H are those where R.sup.21 is
.dbd.CR.sup.aR.sup.b (especially where one of R.sup.a or R.sup.b is
H and the other is CO.sub.2R', and preferably where R' is
C.sub.1-C.sub.10 alkyl, most preferably lower alkyl such as
methyl). Further preferred are those compounds where R.sup.3 is
OH.
[0189] The compounds of Formulae I-V, especially II-V, are
preferred where R.sup.20 is O.sub.2CR' and R' is alkyl (preferably
about C.sub.7-C.sub.20 alkyl), alkenyl (preferably about
C.sub.7-C.sub.20 alkenyl such as
CH.sub.3--CH.sub.2--CH.sub.2--CH.dbd.CH--CH.dbd.CH--) or aryl
(preferably phenyl or naphthyl). Particularly preferred are those
compounds where R.sup.20 is O.sub.2CR' and R.sup.21 is
.dbd.CR.sup.aR.sup.b (especially where one of R.sup.a or R.sup.b is
H and the other is CO.sub.2R', and preferably where R' is
C.sub.1-C.sub.10 alkyl, most preferably lower alkyl such as
methyl). Further preferred are the compounds where R.sup.3 is OH,
R.sup.26 is H and/or Z is --O--.
[0190] The compounds of Formulae I-V, especially II-V, are
preferred where R.sup.21 is .dbd.CR.sup.aR.sup.b (especially where
one of R.sup.a or R.sup.b is H and the other is CO.sub.2R', and
preferably where R' is C.sub.1-C.sub.10 alkyl, most preferably
lower alkyl such as methyl). Further preferred are the compounds
where R.sup.3 is OH, R.sup.26 is H and/or Z is --O--.
[0191] Of the compounds according to Formula I, it is preferred
that L be a group having from about 6 to about 14 carbon atoms.
Also preferred are those compounds where distance "d" (in Formula
Ia) is about 2.5 to 5.0 angstroms, preferably about 3.5 to 4.5
angstroms and most preferably about 4.0 angstroms, such as about
3.92 angstroms. Further preferred are those compounds where L
contains a hydroxyl on the carbon atom corresponding to C3 in the
native bryostatin structure.
[0192] Of the compounds according to Formulae II or III, it is
preferred that X is oxygen. Further preferred are the compounds
where R.sup.3 is OH, R.sup.26 is H and/or Z is --O--.
[0193] Of the compounds according to Formulae II-IV, it is
preferred that R.sup.3 is OH, and especially preferred that X is
oxygen in the case of Formulae II and III. Further preferred are
the compounds where R.sup.26 is H and/or Z is --O--.
[0194] Of the compounds according to Formula II, it is preferred
that R.sup.20 is O.sub.2CR' where R' is alkyl (preferably about
C.sub.7-C.sub.20 alkyl), alkenyl (preferably about C.sub.7-C.sub.20
alkenyl such as
CH.sub.3--CH.sub.2--CH.sub.2--CH.dbd.CH--CH.dbd.CH--) or aryl
(preferably phenyl or naphthyl). Of these, further preferred are
the compounds where R.sup.21 is .dbd.CR.sup.aR.sup.b (especially
where one of R.sup.a or R.sup.b is H and the other is CO.sub.2R',
and preferably where R' is C.sub.1-C.sub.10 alkyl, most preferably
lower alkyl such as methyl). Also preferred are those compounds
where R.sup.3 is OH, R.sup.26 is H and/or Z is --O--.
[0195] Of the compounds according to Formula III, it is preferred
that R.sup.9 is H. It is further preferred that R.sup.8 is H, alkyl
(especially t-butyl), aralkyl,
--CH.sub.2(CH.sub.3).sub.2--CH.sub.2--O--R [particularly where R is
COCH.sub.2Cl, COt-Bu, 2,4,6-trichlorobenzoate, or myristate] or
--(CH.sub.2).sub.nO(O)CR' [particularly where R' is alkyl], R.sup.6
optionally being .dbd.O. Further preferred are those compounds
where R.sup.3 is OH, R.sup.26 is H and/or Z is --O--.
[0196] The compounds according to Formula 12G, particularly where
R.sup.26 is H or C.sub.1-C.sub.6 alkyl.
[0197] The compounds according to Formula 13A, particularly where
R.sup.26 is H or C.sub.1-C.sub.6 alkyl.
[0198] The compounds according to Formula 13B, particularly where
R.sup.26 is H or C.sub.1-C.sub.6 alkyl.
[0199] The compounds according to Formulae 15C, 15D and/or 15E.
[0200] The compounds according to Formula 15F where q is zero.
[0201] The compounds according to Formulae 16H, 16I and/or 16J,
particularly where R.sup.7 is absent.
[0202] The compounds according to Formulae 204, 207, 304 (and the
C26-desmethyl homologue of 304), and 502.
[0203] The compounds according to Formula 705, particularly where
R.sup.26 is H and/or where R.sup.8 and R.sup.9 are H.
[0204] Further preferred are those compounds that combine various
of the above-mentioned features. The single isomers highlighted in
the reaction schemes and examples are also preferred.
[0205] Also preferred (individually, collectively and in any
combination) are the compounds having the structures represented by
the following formulae: ##STR45## ##STR46## ##STR47## ##STR48##
particularly those compounds having one or more of the illustrated
stereochemical configurations. Similarly preferred are the
synthetic processes leading to the above compounds and carrying
those that are intermediates forward.
[0206] Presently, most preferred is the compound of Formula II
where X is oxygen, R.sup.3 is OH, R.sup.20 is
--O--CO--C.sub.7H.sub.15, R.sup.21 is .dbd.CH--CO.sub.2Me and
R.sup.26 is H.
Utility, Testing and Administration
General Utility
[0207] The compounds of the present invention are useful as
bryostatin-like therapeutic agents, and in pharmaceutical
formulations and methods of treatment employing the same. Other
compounds of the invention are useful a precursors in the synthesis
of such agents. Importantly, in many cases, the compounds of the
present invention can be readily synthesized on a large scale, and
thus can be made readily available for commercial purposes as
compared to the low yields and environmental problems inherent in
the isolation of bryostatins from natural sources.
[0208] In one aspect, the compounds of the invention find use as
anticancer agents in mammalian subjects. For example,
representative cancer conditions and cell types against which the
compounds of the invention may be useful include melanoma, myeloma,
chronic lymphocytic leukemia (CLL), AIDS-related lymphoma,
non-Hodgkin's lymphoma, colorectal cancer, renal cancer, prostate
cancer, cancers of the head, neck, stomach, esophagus, anus, or
cervix, ovarian cancer, breast cancer, peritoneal cancer, and
non-small cell lung cancer. The compounds appear to operate by a
mechanism distinct from the mechanisms of other anticancer
compounds, and thus can be used synergistically in combination with
other anticancer drugs and therapies to treat cancers via a
multimechanistic approach. The compounds of the invention exhibit
potencies comparable to or better than previous bryostatins against
many human cancer types.
[0209] In another aspect, the compounds of the invention can be
used to strengthen the immune system of a mammalian subject,
wherein a compound of the invention is administered to the subject
in an amount effective to increase one or more components of the
immune system. For example, strengthening of the immune system can
be evidenced by increased levels of T cells, antibody-producing
cells, tumor necrosis factors, interleukins, interferons, and the
like. Effective dosages may be comparable to those for anticancer
uses, and can be optimized with the aid of various immune response
assay protocols such as are known in the art (e.g., see Kraft,
1996; Lind, 1993; U.S. Pat. No. 5,358,711, all incorporated herein
by reference). The compound can be administered prophylactically,
e.g., for subjects who are about to undergo anticancer therapies,
as well as therapeutically, e.g., for subjects suffering from
microbial infection, burn victims, subjects with diabetes, anemia,
radiation treatment, or anticancer chemotherapy. The
immunostimulatory activity of the compounds of the present
invention is unusual among anticancer compounds and provides a dual
benefit for anticancer applications. First, the immunostimulatory
activity allows the compounds of the invention to be used in
greater doses and for longer periods of time than would be possible
for compounds of similar anticancer activity but lacking
immunostimulatory activity. Second, the compounds of the present
invention can offset the immunosuppressive effects of other drugs
or treatment regimens when used in combination therapies.
Additional features of the invention can be further understood from
the following illustrative examples which are not intended to limit
the scope of the invention in any way.
Testing
[0210] In practicing various aspects of the present invention,
compounds in accordance with the invention can be tested for a
biological activity of interest using any assay protocol that is
predictive of activity in vivo. For example, a variety of
convenient assay protocols are available that are generally
predictive of anticancer activity in vivo.
[0211] In one approach, anticancer activity of compounds of the
invention can be assessed using the protein kinase C assay detailed
in Example 5. In this assay, K.sub.i values are determined for
analogues based on competition with radiolabeled phorbol
12,13-dibutyrate for binding to a mixture of PKC isoenzymes. PKC
enzymes are implicated in a variety of cellular responses which may
be involved in the activity of the bryostatins.
[0212] Example 6 describes another protein kinase C assay which can
be used to assess the binding affinities of compounds of the
invention for binding to the C1B domain of PKC.delta.. Although all
PKC isozymes are upregulated immediately after administration of
bryostatin or tumor promoting phorbol esters followed by an
extended down-regulation period, PKC.delta. appears to be protected
against down regulation by bryostatin 1. Overexpression of
PKC.delta. inhibits tumor cell growth and induces cellular
apoptosis, whereas depleting cells of PKC.delta. can cause tumor
promotion. Accordingly, this assay provides useful binding data for
assessing potential anticancer activity.
[0213] Another useful method for assessing anticancer activities of
compounds of the invention involves the multiple-human cancer cell
line screening assays run by the National Cancer Institute (e.g.,
Boyd, 1989). This screening panel, which involves approximately 60
different human cancer cell lines, is a useful indicator of in vivo
antitumor activity for a broad variety of tumor types (Grever et
al., 1992, Monks et al., 1991), such as leukemia, non-small cell
lung, colon, central nervous system (CNS), melanoma, ovarian,
renal, prostate, and breast cancers. Antitumor activities can be
expressed in terms of ED.sub.50 (or GI.sub.50), where ED.sub.50 is
the molar concentration of compound effective to reduce cell growth
by 50%. Compounds with lower ED.sub.50 values tend to have greater
anticancer activities than compounds with higher ED.sub.50 values.
Example 7 describes a P388 murine lymphocytic leukemia cell assay
which measures the ability of compounds of the invention to inhibit
cellular growth.
[0214] Upon the confirmation of a compounds potential activity in
the above in vitro assays, further evaluation is typically
conducted in vivo in laboratory animals, for example, measuring
reduction of lung nodule metastases in mice with B16 melanoma
(e.g., Schuchter et al, 1991). The efficacy of drug combination
chemotherapy can be evaluated, for example, using the human B-CLL
xenograft model in mice (e.g., Mohammad et al, 1996). Ultimately,
the safety and efficacy of compounds of the invention are evaluated
in human clinical trials.
[0215] Experiments conducted in support of the present invention
demonstrate that compounds of the present invention exhibit high
potencies in several anticancer assays, as summarized in the
Examples.
Administration
[0216] The invention includes a method of inhibiting growth, or
proliferation, of a cancer cell, or enhancing the effectiveness of
other drugs. In the method, a cancer cell is contacted with a
bryostatin analogue compound in accordance with the invention in an
amount effective to inhibit growth or proliferation of the cell. In
a broader aspect, the invention includes a method of treating
cancer in a mammalian subject, especially humans. In the method, a
bryostatin analogue compound in accordance with the invention is
administered to the subject in an amount effective to inhibit
growth of the cancer in the patient. Similarly, in the immune
modulation methods of the invention, a compound of the invention is
administered to a subject in need thereof, in an amount
therapeutically effective for bolstering of the immune system
predisposed toward apoptosis
[0217] Compositions and methods of the present invention have
particular utility in the area of human and veterinary
therapeutics. Generally, administered dosages will be effective to
deliver picomolar to micromolar concentrations of the therapeutic
composition to the target site. Typically, nanomolar to micromolar
concentration at the target site should be adequate for many
applications. Appropriate dosages and concentrations will depend on
factors such as the particular compound or compounds being
administered, the site of intended delivery, and the route of
administration, all of which can be derived empirically according
to methods well known in the art.
[0218] Administration of compounds of the invention in an
appropriate pharmaceutical form can be carried out by any
appropriate mode of administration. Thus, administration can be,
for example, intravenous, topical, subcutaneous, transocular
transcutaneous, intramuscular, oral, intra-joint, parenteral,
peritoneal, intranasal, or by inhalation. The formulations may take
the form of solid, semi-solid, lyophilized powder, or liquid dosage
forms, such as, for example, tablets, pills, capsules, powders,
sustained-release formulations, solutions, suspensions, emulsions,
suppositories, retention enemas, creams, ointments, lotions,
aerosols, and the like. In one embodiment, the formulation has a
unit dosage form suitable for administration of a precise dose.
[0219] Pharmaceutical compositions of the invention typically
include a conventional pharmaceutical carrier or excipient and may
additionally include other medicinal agents, carriers, adjuvants,
antioxidants, and the like. In one embodiment, the composition may
comprise from about 1% to about 75% by weight of one or more
compounds of the invention, with the remainder consisting of
suitable pharmaceutical excipients. For oral administration, such
excipients include pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, talcum, cellulose,
glucose, gelatin, sucrose, magnesium carbonate, for example.
Appropriate excipients can be tailored to the particular
composition and route of administration by methods well known in
the art, e.g., (Gennaro, 1990). Additional guidance for
formulations and methods of administration can be found in patent
references concerning previously known bryostatins, such as U.S.
Pat. Nos. 4,560,774 and 4,611,066 to Pettit et al., which are
incorporated herein by reference.
[0220] Usually, for oral administration, the compositions will take
the form of a pill, tablet or capsule. Thus the composition will
contain, along with active drug, a diluent such as lactose,
sucrose, dicalcium phosphate, and/or other material, a disintegrant
such as starch or derivatives thereof; a lubricant such as
magnesium stearate and the like; and a binder such a starch, gum
acacia, polyvinylpyrrolidone, gelatin, cellulose and/or derivatives
thereof.
[0221] The compounds of the invention may also be formulated into a
suppository comprising, for example, about 0.5% to about 50% of a
compound of the invention, disposed in a polyethylene glycol (PEG)
carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]).
[0222] Liquid compositions can be prepared by dissolving or
dispersing compound (e.g., from about 0.5% to about 20% of final
volume), and optional pharmaceutical adjuvants in a carrier, such
as, for example, aqueous saline, aqueous dextrose, glycerol,
ethanol and the like, to form a solution or suspension. Useful
vehicles also include polyoxyethylene sorbitan fatty acid
monoesters, such as TWEEN.TM. 80, and polyethoxylated castor oils,
such as Cremophor EL.TM. available from BASF (Wyandotte, Md.), as
discussed in PCT Publ. No. WO 97/23208 (which is incorporated
herein by reference), which can be diluted into conventional saline
solutions for intravenous administration. Such liquid compositions
are useful for intravenous administration. One such formulation is
PET diluent which is a 60/30/10 v/v/v mixture of PEG 400,
dehydrated ethanol, and TWEEN.TM.-80. Liquid compositions may also
be formulated as retention enemas.
[0223] The compounds of the invention may also be formulated as
liposomes using liposome preparation methods known in the art.
Preferably, the liposomes are formulated either as small
unilamellar vesicles or as larger vesicles.
[0224] If desired, the composition to be administered may also
contain minor amounts of non-toxic auxiliary substances such as
wetting or emulsifying agents, pH buffering agents, such as, for
example, sodium acetate, sorbitan monolaurate, triethanolamine
oleate, and antioxidants.
[0225] For topical administration, the composition is administered
in any suitable format, such as a lotion or a transdermal patch.
For delivery by inhalation, the composition can be delivered as a
dry powder (e.g., Inhale Therapeutics) or in liquid form via a
nebulizer.
[0226] Methods for preparing such dosage forms are known or will be
apparent to those skilled in the art; for example, see Gennaro
(1990). The composition to be administered will, in any event,
contain a quantity of one or more compounds of the invention in a
pharmaceutically effective amount for relief of the condition being
treated.
[0227] The compounds of the invention may also be introduced in a
controlled-release form, for long-term delivery of drug to a
selected site over a period of several days or weeks. In this case,
the compound of the invention is incorporated into an implantation
device or matrix for delayed or controlled release from the
device.
[0228] The compounds can be incorporated in a biodegradable
material, such as a biodegradable molded article or sponge.
Exemplary biodegradable materials include matrices of collagen,
polylactic acid-polyglycolic acid, and the like. In preparing
bryostatin compounds in matrix form, the compounds may be mixed
with matrix precursor, which is then crosslinked by covalent or
non-covalent means to form the desired matrix. Alternatively, the
compound can be diffused into a preformed matrix. Examples of
suitable materials for use as polymeric delivery systems have been
described e.g., Aprahamian, 1986; Emmanuel, 1987; Friendenstein,
1982; and Uchida, 1987.
[0229] Generally, compounds of the invention are administered in a
therapeutically effective amount, i.e., a dosage sufficient to
effect treatment, which may vary depending on the individual and
condition being treated. Typically, a therapeutically effective
daily dose is from 0.1 .mu.g/kg to 100 mg/kg of body weight per day
of drug. Given the high therapeutic activities of the compounds of
the invention, daily dosages of from about 1 .mu.g/kg and about 1
mg/kg of body weight may be adequate, although dosages greater than
or less than this range can also be used.
[0230] It will be appreciated that the compounds of the invention
may be administered in combination (i.e., together in the same
formulation or in separate formulations administered by the same or
different routes) with any other anti-cancer regimen deemed
appropriate for the patient. For example, the compounds of the
invention may be used in combination with other anticancer drugs
such as vincristine, cisplatin, ara-C, taxanes, edatrexate,
L-buthionine sulfoxide, tiazofurin, gallium nitrate, doxorubicin,
etoposide, podophyllotoxins, cyclophosphamide, camptothecins,
dolastatin, and auristatin-PE, for example, and may also be used in
combination with radiation therapy. In a preferred embodiment, the
combination therapy entails co-administration of an agent selected
from: ara-C, taxol, cisplatin and vincristine.
EXAMPLES
[0231] General Techniques. Unless noted otherwise, materials were
obtained from commercially available sources and used without
further purification. Tetrahydrofuran (THF) and diethyl ether
(Et.sub.2O) were distilled from sodium benzophenone ketyl under a
nitrogen atmosphere. Benzene, dichloromethane (CH.sub.2Cl.sub.2),
acetonitrile (CH.sub.3CN), triethylamine (Et.sub.3N) and pyridine
were distilled from calcium hydride under a nitrogen atmosphere.
Chloroform (CHCl.sub.3), carbon tetrachloride (CCl.sub.4) and
deuterated NMR solvents were dried over 1/16'' bead 4 .ANG.
molecular sieves.
[0232] All operations involving moisture-sensitive materials were
conducted in oven- and/or flame-dried glassware under an atmosphere
of anhydrous nitrogen. Hygroscopic solvents and liquid reagents
were transferred using dry Gastight.TM. syringes or cannulating
needles. In cases where rigorous exclusion of dissolved oxygen was
required, solvents were degassed via consecutive freeze, pump, thaw
cycles or inert gas purge.
[0233] Nuclear magnetic resonance (NMR) spectra were recorded on
either a Varian UNITY INOVA-500, XL-400 or Gemini-300 magnetic
resonance spectrometer. .sup.1H chemical shifts are given in parts
per million (.delta.) downfield from tetramethylsilane (TMS) using
the residual solvent signal (CHCl.sub.3=.delta. 7.27,
benzene=.delta. 7.15, acetone=.delta. 2.04) as internal standard.
Proton (.sup.1H) NMR information is tabulated in the following
format: number of protons, multiplicity (s, singlet; d, doublet; t,
triplet; q, quartet; sept, septet, m, multiplet), coupling
constant(s) (J) in hertz and, in cases where mixtures are present,
assignment as the major or minor isomer, if possible. The prefix
app is occasionally applied in cases where the true signal
multiplicity was unresolved and br indicates the signal in question
was broadened. Proton decoupled .sup.13C NMR spectra are reported
in ppm (.delta.) relative to residual CHCl.sub.3 (.delta. 77.25)
unless noted otherwise.
[0234] Infrared spectra were recorded on a Perkin-Elmer 1600 series
FTIR using samples prepared as thin films between salt plates.
High-resolution mass spectra (HRMS) were recorded at the NIH
regional mass spectrometry facility at the University of
California, San Francisco. Fast Atom Bombardment (FAB)
high-resolution mass spectra were recorded at the University of
California, Riverside. Combustion analyses were performed by Desert
Analytics, Tucson, Ariz., 85719 and optical rotations were measured
on a Jasco DIP-1000 digital polarimeter.
[0235] Flash chromatography was performed using E. Merck silica gel
60 (240-400 mesh) according to the protocol of Still et al. (1978).
Thin layer chromatography was performed using precoated plates
purchased from E. Merck (silica gel 60 PF254, 0.25 mm) that were
visualized using either a p-anisaldehyde or Ce(IV) stain.
[0236] For binding and cell-inhibition studies, dilutions of
bryostatin and bryostatin analogues were performed in glass rather
than plastic, to avoid problems associated with adsorption to
plastic.
Example 1
Exemplary Precursors
1A. Protected Diol Aldehyde 102
[0237] ##STR49##
[0238] Benzyl bromide (7.0 mL, 57.7 mmol) and freshly prepared
Ag.sub.2O (11.0 g, 48.1 mmol) were added successively to an
Et.sub.2O (150 mL) solution of R-(+)-methyl lactate (5.0 g, 48.1
mmol) at rt (room temperature). The resulting suspension was
brought to reflux and stirred for 2 h. The reaction was cooled to
rt, filtered through a pad of Celite.TM. and concentrated in vacuo.
Chromatography on silica gel (10% EtOAc/hexanes) afforded 7.5 g
(80%) of benzyl ether A1 as a colorless oil:
[0239] A1: R.sub.f (15% EtOAc/hexanes)=0.66; IR 2988, 2952, 2874,
1750, 1497, 1454, 1372, 1275, 1207, 1143, 1066, 1025, 739, 698
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.44 (3H, d,
J=6.8 Hz, C27), 3.75 (3H, s, CH.sub.3O), 4.07 (1H, q, J=6.8 Hz,
C26), 4.45 (1H, d, J=11.7 Hz, CH.sub.2Ph), 4.69 (1H, d, J=11.7 Hz,
CH.sub.2Ph), 7.28-7.37 (5H, m, Ph); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 18.6, 51.8, 71.9, 73.9, 127.7, 127.8, 128.3,
137.4, 173.6; HRMS Calcd for C.sub.11H.sub.14O.sub.3 (M.sup.+):
194.0943. Found: 194.0942. ##STR50##
[0240] To a solution of methyl ester A1 (6.3 g, 32.3 mmol) in
Et.sub.2O (150 mL) was added DIBAL-H (1.0M in hexanes, 38.75 mL)
dropwise at -78.degree. C. via cannulating needle. After 5 min at
-78.degree. C., the reaction was quenched with H.sub.2O and
gradually warmed to rt. The resultant thick emulsion was filtered
through a pad of Celite.TM. and sand, rinsing thoroughly with
Et.sub.2O and EtOAc. The organic phase was washed with NaHCO.sub.3
(2.times.), dried over MgSO.sub.4 and concentrated in vacuo to
afford crude aldehyde A2 (not shown) as a light yellow liquid.
[0241] To a solution of SnCl.sub.4 (1.0M in CH.sub.2Cl.sub.2, 32.3
mmol) in CH.sub.2Cl.sub.2 (120 mL) was slowly added a
CH.sub.2Cl.sub.2 solution of aldehyde A2 at -78.degree. C. The
mixture was stirred for an additional 10 min before
allyltrimethylsilane (5.65 mL, 35.53 mmol) was added via syringe.
The ensuing white suspension was kept at -78.degree. C. for 10 min,
quenched by addition of H.sub.2O and allowed to warm to rt. The
aqueous layer was extracted with Et.sub.2O and the combined
organics were dried over MgSO.sub.4 and concentrated in vacuo.
Chromatography on silica gel (10% EtOAc/hexanes) afforded 4.87 g
(76%) of desired diastereomer A3 plus 300 mg (5%) of a putative
mixture.
[0242] A3: R.sub.f (15% EtOAc/hexanes)=0.44; IR (film) 3454, 3066,
3030, 2976, 2871, 1641, 1497, 1554, 1375, 1071, 1028, 992, 914,
737, 698 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.20
(3H, d, J=6.2 Hz, C27), 2.22 (1H, m, C24), 2.35 (1H, m, C24), 2.56
(1H, br s, OH), 3.45 (1H, m, C25), 4.25 (1H, m, C26), 4.44 (1H, d,
J=11.5 Hz, CH.sub.2Ph), 4.66 (1H, d, J=11.5 Hz, CH.sub.2Ph), 5.10
(2H, m, CH.sub.2.dbd.CH), 5.87 (1H, m, CH.sub.2.dbd.CH), 7.28-7.35
(5H, m, Ph); .sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. 15.3, 37.4,
70.9, 74.1, 77.3, 117.03, 127.6, 127.7, 128.3, 134.7, 138.2; HRMS
Calcd for C.sub.13H.sub.18O.sub.2 (M.sup.+): 206.1307. Found:
206.1313. ##STR51##
[0243] To a suspension of potassium tert-butoxide (4.0 g, 35.7
mmol) in 120 mL anhydrous THF was added a solution of alcohol A3
(in 30 mL of THF) slowly over 15 min at 0.degree. C. When complete,
the mixture was stirred at rt for 45 min and then warmed to
60.degree. C. for an additional 30 min. p-Methoxybenzylchloride
(3.56 mL, 26 mmol) was added and the mixture was stirred at
60.degree. C. for 4 h. The reaction was cooled to rt and quenched
with sat. NH.sub.4Cl. The aqueous layer was extracted with
Et.sub.2O (3.times.) and the combined organics were dried over
MgSO.sub.4 and concentrated in vacuo. The crude product was
purified by flash chromatography (5% EtOAc/hexanes) to provide 6.80
g (88%) of differentially protected diol A4 as a colorless oil.
[0244] A4: R.sub.f (15% EtOAc/hexanes)=0.59; IR (film) 3065, 2935,
2868, 1641, 1613, 1586, 1514, 1464, 1380, 1302, 1248, 1094, 1037,
913, 821, 737 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
1.17 (3H, d, J=6.3 Hz, C27), 2.27 (1H, m, C24), 2.40 (1H, m, C24),
3.44 (1H, ddd, J=7.7, 4.7, 4.6 Hz, C25), 3.62 (1H, dq, J=7.7, 6.3
Hz, C26), 3.78 (3H, s, CH.sub.3O), 4.51 (1H, d, J=11.9 Hz,
CH.sub.2Ph), 4.52 (2H, s, CH.sub.2Ph), 4.60 (1H, d, J=11.9 Hz,
CH.sub.2Ph), 5.04 (2H, m, CH.sub.2.dbd.CH), 5.84 (1H, m,
CH.dbd.CH.sub.2), 6.83 (2H, d, J=8.7 Hz, Ar), 7.23-7.33 (7H, m,
Ar); .sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. 15.0, 34.4, 55.1,
71.2, 72.1, 75.7, 80.9, 113.6, 116.6, 127.4, 127.6, 127.6, 128.3,
129.4, 130.9, 135.6, 138.9, 159.2; HRMS Calcd for
C.sub.21H.sub.26O.sub.3 (M.sup.+): 326.1882. Found: 326.1876; Anal.
Calcd for C.sub.21H.sub.26O.sub.3: C, 77.27; H, 8.03. Found: C,
77.22; H, 8.16; [.alpha.].sub.D.sup.20=-8.9.degree. (c 1.43,
CH.sub.2Cl.sub.2).
Formula 102
[0245] A4 (3.0 g, 9.2 mmol) was dissolved in 90 mL
CH.sub.2Cl.sub.2/22.5 mL MeOH and cooled to -78.degree. C. Ozone
was bubbled through the solution which was carefully monitored for
the disappearance of starting material by TLC (thin layer
chromatography). When the consumption of A4 was judged complete,
the system was immediately purged with N.sub.2 for 20 min and
treated with solid thiourea (840 mg, 11.04 mmol). The reaction was
warmed to rt slowly over 5 hours and stirred at rt for 6 hours. The
solvents were removed in vacuo, and the crude mixture was purified
by flash chromatography (20% EtOAc/hexanes) to afford 2.40 g (80%)
of aldehyde 102 as a colorless oil.
[0246] 102: R.sub.f (15% EtOAc/hexanes)=0.31; IR (film) 2868, 2729,
1723, 1612, 1513, 1458, 1384, 1303, 1249, 1174, 1094, 822, 742
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.17 (3H, d,
J=6.4 Hz, C27), 2.57 (1H, ddd, J=16.6, 7.8, 2.4 Hz, C24), 2.67 (1H,
ddd, J=16.6, 4.4, 1.6 Hz, C24), 3.71 (1H, dq, J=6.4, 4.5 Hz, C26),
3.78 (3H, s, CH.sub.3O), 4.05 (1H, ddd, J=7.8, 4.5, 4.4 Hz, C25),
4.45 (1H, d, J=11.8 Hz, CH.sub.2Ph), 4.50 (2H, s, CH.sub.2Ph), 4.58
(1H, d, J=11.8 Hz, CH, Ph), 6.84 (2H, d, J=8.7 Hz, Ar), 7.20-7.35
(7H, m, Ar), 9.70 (1H, dd, J=2.4, 1.6 Hz, CHO); .sup.13C NMR (75
MHz, CDCl.sub.3) .delta. 14.2, 44.1, 55.1, 70.9, 72.0, 74.5, 75.1,
113.8, 127.7, 127.7, 128.4, 129.5, 130.2, 138.4, 159.4, 201.4; HRMS
Calcd for C.sub.20H.sub.24O.sub.4 (M.sup.+): 328.1675. Found:
328.1664; Anal. Calcd for C.sub.20H.sub.24O.sub.4: C, 73.13; H,
7.37. Found: C, 72.81; H, 7.40; [.alpha.].sub.D.sup.20=-9.1.degree.
(c 1.06, CH.sub.2Cl.sub.2).
1B. Diketone 101
[0247] ##STR52##
[0248] A mixture of methylisopropyl ketone (53.4 mL, 0.5 mol) and
paraformaldehyde (19.5 g, 0.65 mol) in 200 mL CF.sub.3CO.sub.2H was
stirred at 60.degree. C. for 18 h. The reaction mixture was
concentrated on a rotary evaporator (warm water bath) with a KOH
trap and poured into a cold (5.degree. C.) mixture of EtOAc and
sat. aqueous NaHCO.sub.3. The layers were separated and the organic
layer was washed with brine, dried over MgSO.sub.4, and
concentrated in vacuo. Short path distillation gave the
trifluoroacetate ester of 3,3-dimethyl-4-hydroxy butanone
(bp=53-55.degree. C. at 2 mm Hg, 72.4 g) in 68% yield. This
material was dissolved in 400 mL MeOH and treated with 190 mL of 2N
NaOH at 0.degree. C. After 1 h at 0.degree. C., the reaction
mixture was concentrated in vacuo and the residue was partitioned
between EtOAc and sat. aqueous NH.sub.4Cl. The organic layer was
washed with brine, dried over MgSO.sub.4, and concentrated to
afford 40.1 g (.about.100%) of 3,3-dimethyl-4-hydroxy butanone (A5)
as a colorless liquid. Crude A5 was taken up in 200 mL anhydrous
DMF and treated with t-butyldimethylsilylchloride (57.3 g, 0.38
mol) and imidazole (25.9 g, 0.38 mol) at 0.degree. C. After
stirring at rt for 2 h, the solution was diluted with EtOAc, washed
with sat. aqueous NaHCO.sub.3 solution and brine, dried over
MgSO.sub.4 and concentrated in vacuo. The residue was distilled
under reduced pressure to afford silyl ether ketone A6
(bp=65.degree. C. at 1.5 mm Hg, 55.76 g, 81%) as a colorless
oil.
[0249] A6: R.sub.f (25% EtOAc/hexanes)=0.90; IR (film) 2957, 2931,
2858, 1713, 1473, 1362, 1257, 1136, 1097, 838, 777 cm.sup.-1;
.sup.1H NMR (300 MHZ, CDCl.sub.3) .delta. 0.03 (6H, s), 0.87 (9H,
s), 1.09 (6H, s), 2.17 (3H, s), 3.57 (2H, s); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 5.7, 18.1, 21.4, 25.7, 26.1, 49.6, 70.2, 213.4;
HRMS Calcd for C.sub.12H.sub.26O.sub.2Si (M.sup.+-CH.sub.3):
215.1467. Found: 215.1469. ##STR53##
[0250] To a stirred solution of diisopropylamine (12.6 mL, 96 mmol)
in THF (200 mL) was added n-butyllithium (38.4 mL, 2.5M in hexane,
96 mmol) dropwise at 0.degree. C. The mixture was stirred at
0.degree. C. for 30 min, cooled to -78.degree. C., and treated with
a solution of ketone A6 (20.0 g, 87 mmol) in THF (50 mL) slowly
over 10 min. After stirring at -78.degree. C. for 40 min,
acetaldehyde (5.34 mL, 96 mmol) was added and the mixture was kept
at -78.degree. C. for 2 h and at -40.degree. C. for 1.5 h. The
reaction was quenched with saturated NH.sub.4Cl solution (20 mL)
and allowed to warm gradually to rt. The mixture was extracted with
ether and the combined organics were washed with brine, dried over
MgSO.sub.4 and concentrated in vacuo to afford nearly pure aldol A7
(19 g, 80%) as a pale yellow oil. An analytical sample was obtained
following chromatography on silica gel.
[0251] A7: R.sub.f (15% EtOAc/hexanes)=0.33; IR (film) 2958, 2930,
2858, 1699, 1472, 1392, 1363, 1258, 1101, 838, 777 cm.sup.-1;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 4.16 (1H, m), 3.55 (2H,
s), 3.36 (1H, s), 2.74 (1H, dd, J=18.0, 2.4 Hz), 2.68 (1H, dd,
J=18.0, 9.0 Hz), 1.16 (3H, d, J=6.3 Hz), 1.08 (3H, s), 1.07 (3H,
s), 0.85 (9H, s), 0.01 (6H, s); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. -5.7, 18.1, 21.3, 22.3, 25.8, 46.2, 49.7, 63.9, 70.2,
216.8; Anal. Calcd for C.sub.14H.sub.30O.sub.3Si: C, 61.26; H,
11.02. Found: C, 61.01; H, 11.28.
Formula 101
[0252] To a solution of oxalyl chloride (5.3 mL, 60.4 mmol) in
CH.sub.2Cl.sub.2 (150 mL) was added dimethyl sulfoxide (8.56 mL,
120.8 mmol) dropwise at -78.degree. C. After 20 min, a solution of
crude alcohol A7 (15.0 g, 54.9 mmol) in CH.sub.2Cl.sub.2 (150 mL)
was added over 10 min and the mixture was stirred at -78.degree. C.
for 1 h. Et.sub.3N (38 mL, 275 mmol) was added and the mixture was
stirred for 20 min, brought to 0.degree. C., quenched with sat.
aqueous NH.sub.4Cl and diluted with EtOAc. The layers were
separated and the aqueous layer was extracted with EtOAc
(2.times.). The combined organics were washed with brine, dried
over MgSO.sub.4, and concentrated in vacuo. Flash chromatography on
silica gel (5.fwdarw.10% EtOAc/hexanes) provided enolic
.beta.-diketone 101 (12.0 g, 81%) as a yellow oil.
[0253] 101: R.sub.f(15% EtOAc/hexanes)=0.68; IR (film) 2957, 2930,
1606, 1472, 1362, 1257, 1102, 838, 777 cm.sup.-1; .sup.1H NMR (300
MHZ, CDCl.sub.3) .delta. 0.01 (6H, s), 0.85 (9H, s), 1.10 (6H, s),
2.05 (3H, s), 3.53 (2H, s), 5.62 (1H, s); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. -5.5, 18.3, 22.1, 25.5, 25.9, 44.9, 70.0, 98.0,
192.6, 198.5; Anal. Calcd for C.sub.14H.sub.28O.sub.3Si: C, 61.72;
H, 10.36. Found: C, 61.08; H, 10.43.
1C. Dibenzyl Ether Octanoate 111
Formulae 104a and 104b
[0254] To a solution of diisopropylamine (3.21 mL, 23 mmol) in 25
mL THF at -60.degree. C. was added n-butyllithium (1.6 M in
hexanes, 13.83 mL, 22.13 mmol) dropwise. The colorless solution was
warmed to 0.degree. C. and stirred for 30 min. A THF solution (35
mL) of diketone 101 (Example 1B) (2.98 g, 10.9 mmol) was
subsequently added via cannula and the mixture was stirred for 1 h
at 0.degree. C. The reaction was re-cooled to -78.degree. C. and
treated with a solution of aldehyde 102 (Example 1A) (3.0 g, 9.15
mmol) in 35 mL THF. After 30 min at -78.degree. C., the mixture was
quenched with sat. NH.sub.4Cl and brought to rt. The aqueous layer
was extracted with Et.sub.2O, the combined organics were dried over
MgSO.sub.4 and the solvent was removed in vacuo. The crude residue
was quickly passed through a column of silica gel (20%
EtOAc/hexanes) to provide 5.40 g (98%) of aldol diastereomer
mixture 103 as an approximately 1:1 mixture of diastereomers.
[0255] A portion of isolated 103 (2.50 g, 4.2 mmol) was dissolved
in 60 mL anhydrous toluene and treated with 4 .ANG. molecular
sieves (1.55 g) and p-toluenesulfonic acid (60 mg). The reaction
was stirred at room temperature for 4.5 h, quenched with 2 mL
pyridine and concentrated. The residue was taken up in Et.sub.2O,
washed with sat. NaHCO.sub.3, dried over MgSO.sub.4 and the solvent
was removed in vacuo. Flash chromatography (20->25%
EtOAc/hexanes) afforded pyrone compounds 104a (1.0 g) and 104b (1.2
g) in 90% overall yield as colorless oils.
[0256] 104b: R.sub.f (30% EtOAc/hexanes)=0.59; .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 0.05 (6H, s, TBS), 0.85 (9H, s, TBS), 1.05
(3H, s, C18 Me), 1.06 (3H, s, C18 Me), 1.20 (3H, d, J=5.8 Hz, C27),
1.98 (2H, m, C24), 2.12 (1H, dd, J=16.7, 3.6 Hz, C24), 2.28 (1H,
dd, J=16.7, 13.7 Hz, C22), 3.44 (2H, s, C17), 3.54 (1H, m, C25),
3.72 (1H, m, C26), 3.81 (3H, s, CH.sub.3O), 4.23 (1H, m, C23), 4.40
(1H, d, J=11.2 Hz, CH.sub.2Ph), 4.47 (1H, d, J=11.8 Hz,
CH.sub.2Ph), 4.52 (1H, d, J=11.2 Hz, CH.sub.2Ph), 4.63 (1H, d,
J=11.8 Hz, CH.sub.2Ph), 5.39 (1H, s, C20), 6.85 (2H, d, J=8.7 Hz,
Ar), 7.17-7.35 (7H, m, Ar); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. -5.5, 14.7, 18.2, 22.4, 25.9, 34.5, 40.9, 42.4, 55.4, 69.6,
71.2, 72.0, 74.3, 76.2, 103.5, 114.1, 127.9, 128.0, 128.7, 128.8,
129.9, 130.4, 138.8, 159.7, 182.2, 193.7; HRMS Calcd for
C.sub.34H.sub.50O.sub.6Si (M.sup.+): 582.3377. Found: 582.3370
[0257] 104a: R.sub.f (30% EtOAc/hexanes)=0.52; IR 2955, 2857, 1667,
1599, 1514, 1397, 1336, 1301, 1249, 1174, 1102, 838 cm.sup.-1;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.05 (6H, s, TBS), 0.87
(9H, s, TBS), 1.10 (3H, s, C18 Me), 1.14 (3H, s, C18 Me), 1.21 (3H,
d, J=6.3 Hz, C27), 1.71 (1H, m, C24), 2.10 (1H, m, C24), 2.42 (2H,
m, C22), 3.48 (1H, d, J=9.3 Hz, C17), 3.58 (1H, d, J=9.3 Hz, C17),
3.76 (1H, m, C26), 3.82 (3H, s, CH.sub.3O), 3.83 (1H, m, C25), 4.43
(1H, d, J=10.7 Hz, CH.sub.2Ph), 4.56 (1H, br m, C23), 4.57 (1H, d,
J=11.8 Hz, CH.sub.2Ph), 4.63 (1H, d, J=10.7 Hz, CH.sub.2Ph), 4.65
(1H, d, J=11.8 Hz, CH.sub.2Ph), 5.47 (1H, s, C20), 6.87 (2H, d,
J=8.6 Hz, Ar), 7.20 (2H, d, J=8.6 Hz, Ar), 7.32-7.38 (5H, m, Ar);
.sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. -5.6, 14.4, 18.1, 22.3,
22.5, 25.7, 35.2, 41.5, 42.3, 55.2, 69.4, 71.1, 72.8, 74.8, 75.9,
76.4, 103.2, 113.8, 127.5, 128.3, 129.3, 130.3, 138.5, 159.2,
181.1, 193.4; HRMS Calcd for C.sub.34H.sub.50O.sub.6Si (M.sup.+):
582.3377. Found: 582.3369; Anal. Calcd for
C.sub.34H.sub.50O.sub.6Si: C, 70.06; H, 8.65. Found: C, 69.95; H,
8.77; [.alpha.].sub.D.sup.20=+43.9.degree. (c 0.70,
CH.sub.2Cl.sub.2).
Formula 105
[0258] To a solution of pyrone 104a (680 mg, 2.4 mmol) and
CeCl.sub.3-7H.sub.2O (218 mg, 0.59 mmol) in 40 mL methanol was
added solid NaBH.sub.4 (89 mg, 2.3 mmol) in a single portion at
-20.degree. C. The reaction mixture was stirred for 1 h at
-20.degree. C. and then quenched with 50 mL brine. The mixture was
brought to rt, filtered through a pad of Celite.TM. and extracted
with EtOAc (4.times.). The combined organics were dried over
Na.sub.2SO.sub.4 and concentrated in vacuo to afford a crude
allylic alcohol. This moderately stable oil was carried forward
without purification.
[0259] Crude allylic alcohol was dissolved in 45 mL
CH.sub.2Cl.sub.2/MeOH (2:1) and treated with solid NaHCO.sub.3 (243
mg, 2.9 mmol). Purified m-CPBA (377 mg, 2.20 mmol) was added in a
single portion and the reaction mixture was stirred at rt for 1 h.
The mixture was quenched with Et.sub.3N (15 mL), stirred for 20
min, diluted with 200 mL Et.sub.2O, and filtered through a pad of
Celite.TM.. The filtrate was concentrated in vacuo and the residue
was purified by flash chromatography (40% EtOAc/hexanes) to give
650 mg (71% from 104a) of syn diol 105 as a colorless oil.
[0260] 105: R.sub.f (30% EtOAc/hexanes)=0.29; IR (film) 3372, 1613,
1514, 1465, 1390, 1302, 1249, 1180, 1150, 1072, 935, 837, 779, 736,
698, 673 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.09
(6H, s, TBS), 0.91 (9H, s, TBS), 1.03 (3H, s, C18 Me), 1.07 (3H, s,
C18 Me), 1.18 (3H, d, J=6.3 Hz, C27), 1.50-1.68 (2H, m, C24/C22),
1.80 (1H, m, C22), 2.57 (1H, app d, J=11.2 Hz, C24), 3.28 (3H, s,
CH.sub.3O), 3.39, (1H, d, J=10.1 Hz, C17), 3.62 (1H, d, J=11.2 Hz,
C17), 3.79 (3H, s, CH.sub.3O), 3.71-3.94 (5H, m, CH.sub.2Ph,
C23/C25/C26), 4.40 (1H, d, J=10.7 Hz, CH.sub.2Ph), 4.56 (1H, d,
J=11.8 Hz, CH.sub.2Ph), 4.62 (1H, d, J=11.5 Hz, CH.sub.2Ph), 5.40
(1H, d, J=2.5 Hz, OH), 6.84 (2H, d, J=8.7 Hz, PMB), 7.18 (2H, d,
J=8.7 Hz, PMB), 7.37-7.26 (5H, m, Bn).
Formula 106
[0261] A solution of diol 105 (0.81 g, 1.28 mmol) and
4-dimethylaminopyridine (DMAP, 0.55 g, 4.48 mmol) in 22 mL
CH.sub.2Cl.sub.2 was cooled to -10.degree. C. and treated with
benzoyl chloride (193 .mu.L, 1.66 mmol) dropwise via syringe. The
resulting mixture was stirred at -10.degree. C. for 30 min,
quenched with sat NaHCO.sub.3 and diluted with EtOAc (150 mL). The
organic layer was washed with H.sub.2O and brine, dried over
Na.sub.2SO.sub.4 and concentrated in vacuo to afford a crude
mixture of C21 monobenzoate and 4-dimethylaminopyridine as a
colorless paste.
[0262] The crude C21 monobenzoate was taken up in 45 mL
CH.sub.2Cl.sub.2 and treated with solid
1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (Dess-Martin
periodinane or DMP, 1.78 g, 4.20 mmol) at rt. The solution was
stirred for 10 h at rt after which a second portion (0.50 g, 1.18
mmol) of DMP was added. The opaque white mixture was stirred for
another 1.5 h and quenched with 30 mL sat.
NaHCO.sub.3/Na.sub.2S.sub.2O.sub.3. The two phase system was
vigorously stirred until the organic layer cleared (.about.25 min).
The layers were separated and the aqueous phase was extracted with
CH.sub.2Cl.sub.2 (2.times.). The combined organics were dried over
Na.sub.2SO.sub.4 and concentrated in vacuo to provide a colorless
semi-solid. Flash chromatography on silica gel (25% EtOAc/hexanes)
gave 106 (0.85 g-90% from 105) as a colorless oil.
[0263] 106: R.sub.f (15% EtOAc/hexanes)=0.43; IR (film) 2954, 2933,
1754, 1723, 1610, 1513, 1451, 1267, 1251 cm.sup.-1; .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 0.01 (6H, s, TBS), 0.88 (9H, s, TBS),
1.12 (3H, s, C18 Me), 1.14 (3H, s, C18 Me), 1.19 (3H, d, J=6.3 Hz,
C27), 1.64-1.73 (1H, m, C24), 1.82-1.90 (1H, m, C24), 2.13 (1H, app
q, J=12.4 Hz, C22), 2.40 (1H, ddd, J=12.4, 6.5, 1.6 Hz, C22), 3.54
(3H, s, C19 OCH.sub.3), 3.65 (1H, d, J=9.2 Hz, C17), 3.69 (1H, d,
J=9.2 Hz, C17) 3.72-3.81 (2H, m), 3.81 (31H, s, ArOCH.sub.3), 3.89
(1H, m), 4.49 (1H, d, J=10.7 Hz, CH.sub.2Ar), 4.57 (1H, d, J=13.0
Hz, CH.sub.2Ar), 4.61 (1H, d, J=13.0 Hz, CH.sub.2Ar), 4.62 (1H, d,
J=10.7 Hz, CH.sub.12Ar), 5.80 (1H, dd, J=12.9, 6.3 Hz, C21), 6.87
(2H, d, J=8.3 Hz, Ar), 7.26-7.36 (7H, m, Ar), 7.45 (2H, m, Ar),
7.58 (1H, app t, J=7.2 Hz, Ar), 8.09 (2H, d, J=7.2 Hz, Ar);
.sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. -5.3, 14.7, 18.5, 20.4,
20.6, 26.1, 35.5, 40.4, 45.1, 53.8, 55.5, 65.4, 67.2, 71.4, 73.0,
73.3, 75.1, 103.8, 114.1, 127.9, 128.6, 128.7, 129.7, 129.9, 130.2,
131.0, 133.6, 138.9, 159.6, 165.9, 198.6; HRMS Calcd for
C.sub.42H.sub.58O.sub.9Si (M+-MeOH): 702.3588. Found: 702.3563;
Anal. Calcd for C.sub.42H.sub.58O.sub.9Si: C, 68.63; H, 7.96.
Found: C, 68.28; H, 8.11; [.alpha.].sub.D.sup.20=+22.4.degree. (c
1.53, CH.sub.2Cl.sub.2).
Formula 107
[0264] A solution of benzoate 106 (0.85 g, 1.16 mmol) in 20 mL
THF/MeOH (3:1) was titrated with SmI.sub.2 (0.1 M in THF, 25.5 mL,
2.55 mmol) at -78.degree. C. until an olive green color persisted.
The reaction mixture was quenched with 4 mL sat. NaHCO.sub.3,
warmed to rt and diluted with EtOAc (150 mL). The organic layer was
washed with NaHCO.sub.3, H.sub.2O and brine, dried over
Na.sub.2SO.sub.4 and concentrated in vacuo. Flash chromatography on
silica gel (20% EtOAc/hexanes) afforded ketone 107 (675 mg, 95%) as
a light yellow oil.
[0265] 107: R.sub.f (15% EtOAc/hexanes)=0.39; IR (film) 2954, 2930,
1723, 1612, 1514, 1464, 1250, 1088 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 0.01 (6H, s, TBS), 0.87 (9H, s, TBS), 0.95 (3H,
s, C18 Me), 1.05 (3H, s, C18 Me), 1.19 (3H, d, J=6.3 Hz, C27), 1.64
(1H, m, C24), 1.85-1.96 (3H, m, C21+C24), 2.29 (1H, 5 line m, C22),
2.65 (1H, m, C22), 3.23 (3H, s, C19 OCH.sub.3), 3.31 (1H, d, J=9.9
Hz, C17), 3.71 (1H, d, J=9.9 Hz, C17), 3.78 (3H, s, ArOCH.sub.3),
3.79 (1H, m), 3.98 (1H, m), 4.17 (1H, m), 4.43 (1H, d, J=11.0 Hz,
CH.sub.2Ar), 4.58 (2H, s, CH.sub.2Ar), 4.60 (1H, d, J=11.0 Hz,
CH.sub.2Ar), 6.84 (2H, d, J=8.7 Hz, PMB), 7.19 (2H, d, J=8.7 Hz,
PMB), 7.28-7.35 (5H, m, Bn); .sup.13CNMR (75 MHz, CDCl.sub.3)
.delta. -5.5, -5.4, 14.5, 18.6, 19.8, 20.2, 26.0, 29.7, 36.5, 38.2,
46.2, 52.4, 55.4, 68.6, 70.6, 71.3, 72.3, 74.7, 103.6, 114.1,
127.9, 128.7, 129.6, 131.0, 138.9, 159.5, 207.5; HRMS Calcd for
C.sub.35H.sub.54O.sub.7Si (M+-MeOH): 582.3377. Found: 582.3372;
Anal. Calcd for C.sub.35H.sub.54O.sub.7Si: C, 68.37; H, 8.85.
Found: C, 68.04; H, 8.84; [.alpha.].sub.D.sup.20=+21.9.degree. (c
0.72, CH.sub.2Cl.sub.2). ##STR54##
109.1 (Formula 109 where R.sup.21 is .dbd.CH--CO.sub.2Me)
[0266] To a solution of diisopropylamine (150 .mu.L, 1.15 mmol) in
THF (1.61 mL) was added n-BuLi (2.5 M in hexanes, 440 .mu.L, 1.10
mmol) dropwise at 0.degree. C. After 5 min at 0.degree. C., a 1.81
mL aliquot (0.5 M LDA, 0.90 mmol) was removed via syringe and
slowly added to a solution of ketone 107 (483 mg, 0.79 mmol) in THF
(20 mL) at -78.degree. C. The solution was stirred for 10 min,
treated with a stock solution of OHCCO.sub.2Me (0.5 M in Et.sub.2O,
4.0 mL, 2.0 mmol), kept at -78.degree. C. for 20 min and quenched
with 3 mL sat. NH.sub.4Cl. The reaction mixture was brought to rt
and diluted with 200 mL EtOAc. The organic layer was washed with
H.sub.2O (2.times.) and brine, dried over Na.sub.2SO.sub.4 and
concentrated in vacuo. The crude residue was chromatographed on
silica gel (35% EtOAc/hexanes) to afford residual 107 (142 mg) and
aldols 108 as a mixture of diastereomers (352 mg, 90% based on
recovered 107).
[0267] The isolated 108 mixture and Et.sub.3N (418 .mu.L, 3.0 mmol)
were dissolved in anhydrous CH.sub.2Cl.sub.2 (15 mL) and cooled to
-10.degree. C. Methanesulfonylchloride (116 .mu.L, 1.5 mmol) was
added via syringe and the solution was stirred at -10.degree. C.
for 30 min. 5 mL sat. NaHCO.sub.3 was added, the reaction mixture
was warmed to rt and diluted with 100 mL EtOAc. The organic layer
was washed with H.sub.2O and brine, dried over Na.sub.2SO.sub.4 and
concentrated in vacuo. The residue was immediately dissolved in THF
(30 mL) and treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU,
75 .mu.L, 0.50 mmol) dropwise at rt. The resulting bright yellow
solution was stirred at rt for 20 min, treated with sat. NH.sub.4Cl
and diluted with 150 mL EtOAc. The organic layer was washed with
H.sub.2O and brine, dried over Na.sub.2SO.sub.4 and concentrated in
vacuo to afford an orange residue which was chromatographed on
silica gel (20% EtOAc/hexanes) to afford exocyclic methacrylate
(enone) 109.1 (267 mg, 78% from 108) as a yellow oil.
[0268] 109.1: R.sub.f (30% EtOAc/hexanes)=0.63; IR (film) 2954,
2930, 1724, 1707, 1612, 1514, 1464, 1250, 1088 cm.sup.-1; .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 0.05 (6H, s, TBS), 0.81 (9H, s,
TBS), 0.91 (3H, s, C18 Me), 1.00 (3H, s, C18 Me), 1.19 (3H, d,
J=6.4 Hz, C27), 1.74 (1H, m, C24), 1.94 (1H, m, C24), 2.71 (1H,
ddd, J=17.6, 12.4, 3.1 Hz, C22), 3.19 (3H, s, C19 OCH.sub.3), 3.26
(1H, d, J=10.0 Hz, C17), 3.49 (1H, d, J=17.6 Hz, C22), 3.68 (1H, d,
J=10.0 Hz, C17), 3.74 (3H, s), 3.77 (3H, s), 3.80 (1H, m), 3.97
(1H, m), 4.19 (1H, m), 4.40 (1H, d, J=10.9 Hz, CH.sub.2Ar),
4.53-4.63 (3H, m, CH.sub.2Ar), 6.64 (1H, d, J=1.9 Hz, C34), 6.82
(2H, d, J=8.7 Hz, PMB), 7.14 (2H, d, J=8.7 Hz, PMB), 7.27-7.36 (5H,
m, Bn); .sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. -5.9, -5.8, 14.1,
18.3, 19.3, 19.8, 25.7, 35.2, 36.0, 46.6, 51.6, 52.1, 55.1, 68.2,
69.5, 71.0, 71.7, 74.2, 76.0, 104.0, 113.8, 122.4, 127.6, 128.4,
129.1, 130.6, 138.6, 147.3, 159.2, 166.4, 196.2; HRMS Calcd for
C.sub.38H.sub.56O.sub.9Si (M+-MeOH): 652.3433. Found: 652.3435;
Anal. Calcd for C.sub.38H.sub.56O.sub.9Si: C, 66.64; H, 8.24.
Found: C, 66.87; H, 8.13; [.alpha.].sub.D.sup.20=-55.8.degree. (c
0.78, CH.sub.2Cl.sub.2). ##STR55##
111.1 (Formula 111 where R.sup.20 is --O--CO--C.sub.7H.sub.15 and
R.sup.21 is .dbd.CH--CO.sub.2Me)
[0269] To a solution of enone 109.1 (502 mg, 0.734 mmol) and
CeCl.sub.3-7H.sub.2O (137 mg, 0.367 mmol) in methanol (23 mL) was
added solid NaBH.sub.4 (56 mg, 1.47 mmol) in a single portion at
-30.degree. C. Rapid gas evolution subsided after 3 min. After an
additional 30 min at -30.degree. C., the reaction mixture was
poured directly onto a silica gel column and the product quickly
eluted with 25% EtOAc/hexanes to afford the corresponding axial
alcohol 110.1 (478 mg) as a colorless oil.
[0270] Octanoic acid (232 mg, 1.61 mmol) and Et.sub.3N (292 .mu.L,
2.20 mmol) were dissolved in 20 mL toluene and treated with
2,4,6-trichlorobenzoylchloride (230 .mu.L, 1.47 mmol) dropwise at
rt. After 1 h at rt, a toluene solution (7 mL) of freshly prepared
110.1 was added gradually via syringe and stirring was continued
for 40 min. The reaction mixture was quenched with 10 mL sat.
NaHCO.sub.3, diluted with EtOAc and washed successively with sat.
NH.sub.4Cl and brine. The organics were dried over
Na.sub.2SO.sub.4, the solvent was removed in vacuo, and the residue
was chromatographed on silica gel (25% EtOAc/hexanes) to provide
octanoate 111.1 as a colorless oil (551 mg, 93% from 109.1).
[0271] 111.1: R.sub.f (25% Et.sub.2O/hexanes)=0.33; IR (film) 2928,
2857, 1747, 1722, 1667, 1614, 1514, 1463, 1250, 1155, 1081, 836.2
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. -0.01 (6H, s,
TBS), 0.88 (12H, br s, TBS+octanoate Me), 0.99 (3H, s, C18 Me),
1.03 (3H, s, C18 Me), 1.19 (3H, d, J=6.3 Hz, C27), 1.20-1.35 (8H,
m), 1.60-1.80 (3H, m), 1.89 (1H, m, C24), 2.31-2.40 (3H, m), 3.26
(3H, s, C19 OCH.sub.3), 3.44 (1H, dd, J=15.6, 1.8 Hz, C22), 3.56
(1H, d, J=9.3 Hz, C17), 3.60 (1H, d, J=9.3 Hz, C17), 3.68 (3H, s),
3.78 (1H, m), 3.79 (3H, s), 3.93 (1H, dd, J=8.4, 4.8 Hz), 4.13 (1H,
m), 4.38 (1H, d, J=10.8 Hz, CH.sub.2Ar), 4.57 (1H, d, J=10.8 Hz,
CH.sub.2Ar), 4.60 (2H, s, CH.sub.2Ar), 5.57 (1H, s, C20), 5.89 (1H,
s, C34), 6.83 (2H, d, J=8.4 Hz, PMB), 7.16 (2H, d, J=8.4 Hz, PMB),
7.28-7.38 (5H, m, Bn); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
-5.4, 14.1, 14.2, 14.4, 18.4, 20.7, 22.7, 24.8, 26.0, 28.9, 26.0,
28.9, 31.7, 33.2, 34.6, 36.4, 47.1, 51.2, 55.3, 67.6, 68.1, 71.2,
72.2, 74.7, 76.5, 103.1, 114.0, 117.0, 127.8, 128.6, 129.5, 129.9,
130.9, 139.0, 153.1, 159.5, 166.7, 172.2; HRMS Calcd for
C.sub.46H.sub.72O.sub.10Si (M+-MeOH): 780.4632. Found: 780.4610;
[.alpha.].sub.D.sup.20=-5.1.degree. (c 1.80, CH.sub.2Cl.sub.2).
Example 2
Exemplary Linkers
2A. Ketal Acid 406
Formula 402
[0272] To a stirred solution of the 1,3 menthone acetal of
1,3,5-pentanetriol 401 (3.33 g, 13 mmol) prepared by the method of
Harada et al. (1993) in 23 mL of anhydrous CH.sub.2Cl.sub.2 was
added 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one
(Dess-Martin periodinane, 6.60 g, 15.6 mmol) in a single portion.
The mixture was stirred at rt for 30 min, poured onto a column of
silica gel and the product eluted with 15% EtOAc/hexanes to afford
3.013 g (90%) of pure aldehyde 402 as a colorless oil.
[0273] 402: R.sub.f (20% EtOAc/hexanes)=0.50; IR (film) 2952, 2869,
1728, 1456, 1383, 1308, 1265 cm.sup.-; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 9.80 (1H, dd, J=1.8, 2.5 Hz), 4.36 (1H, dddd,
J=2.9, 4.3, 7.4, 8.2 Hz), 4.13 (1H, ddd, J=2.7, 11.7, 11.9 Hz),
3.83 (1H, ddd, J=1.3, 5.2, 11.7 Hz), 2.72 (1H, ddd, J=1.9, 3.1,
13.5 Hz), 2.56 (1H, ddd, J=2.5, 8.2, 16.1 Hz), 2.44 (1H, ddd,
J=1.8, 4.3, 16.1 Hz), 2.39 (1H, dsept, J=1.6, 7.1 Hz), 1.54-1.76
(3H, m), 1.29-1.53 (4H, m), 1.17 (1H, ddd, J=1.9, 4.1, 12.4 Hz),
0.90 (3H, d, J=6.3 Hz), 0.87 (3H, d, J=7.0 Hz), 0.85 (3H, d, J=7.0
Hz), 0.72 (1H, t, J=13.2 Hz); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. 201.4, 100.9, 63.8, 58.7, 51.1, 49.9, 37.2, 34.8, 31.1,
28.9, 24.2, 23.7, 22.2, 21.7, 18.8; HRMS Calc'd for
C.sub.15H.sub.26O.sub.3: 254.1882. Found: 254.1877;
[.alpha.].sub.D.sup.20-11.2.degree. (c 1.28, CHCl.sub.3).
Formulae 403a and 403b
[0274] To a stirred solution of aldehyde 402 (3.013 g, 11.86 mmol)
in 40 mL of anhydrous CH.sub.2Cl.sub.2 was added a solution of
(+)-Eu(hfc).sub.3 (1.42 g, 1.19 mmol) in CH.sub.2Cl.sub.2 (16 mL)
at rt. The resultant clear yellow solution was stirred for 5 min
before 1-methoxy-3-(trimethylsilyloxy)-1,3-butadiene (3.47 mL, 3.07
g, 17.8 mmol) was introduced via syringe. The yellow solution was
stirred at rt for 20 h, treated with 0.5 mL CF.sub.3CO.sub.2H and
stirred for an additional 15 min. The solution was quenched with
sat. NaHCO.sub.3, diluted with EtOAc (200 mL), washed with H.sub.2O
and brine, dried over MgSO.sub.4 and concentrated in vacuo.
Purification by flash chromatography (20->25% EtOAc/hexanes)
provided anti pyrone 403b (2.53 g, 66%) and syn pyrone 403a (1.30
g, 34%) as colorless solids.
[0275] 403b: mp=113-114.degree. C. (hexanes); R.sub.f (15%
EtOAc/hexanes)=0.25; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.7.31
(1H, d, J=6.0 Hz), 5.40 (1H, d, J=6.0 Hz), 4.67 (1H, dddd, J=3.0,
5.8, 9.6, 11.8 Hz), 4.03-4.15 (2H, m), 3.81 (1H, ddd, J=1.2, 5.2,
11.6 Hz), 2.70 (1H, ddd, J=1.9, 2.8, 13.5 Hz), 2.42-2.56 (2H, m),
2.38 (1H, dsept, J=1.6, 6.9 Hz), 1.91 (1H, ddd, J=2.3, 9.6, 14.3
Hz), 1.28-1.75 (8H, m), 1.18 (1H, ddd, J=1.9, 3.9, 12.5 Hz), 0.88
(3H, d, J=6.6 Hz), 0.87 (3H, d, J=7.1 Hz), 0.83 (3H, d, J=6.9 Hz),
0.70 (1H, t, J=12.6 Hz); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
192.7, 162.9, 107.3, 100.7, 75.7, 63.3, 58.9, 51.1, 42.5, 41.7,
37.3, 34.8, 31.7, 29.0, 24.2, 23.6, 22.2, 21.9, 18.9; LRMS (EI):
322 (68), 307 (22), 265 (33), 237 (67), 153 (24), 191 (94), 139
(52), 112 (20), 97 (100), 83 (31), 81 (47), 71 (24), 69 (35); HRMS
Calcd for C.sub.19H.sub.30O.sub.4: 322.2144. Found: 322.2142; Anal.
Calcd for C.sub.19H.sub.30O.sub.4: C, 70.77; H, 9.38. Found: C,
70.91; H, 9.58; [.alpha.].sub.D.sup.20+42.5.degree. (c 1.59,
CH.sub.2Cl.sub.2).
[0276] 403a: R.sub.f(15% EtOAc/hexanes)=0.15; IR 2952, 2869, 1682,
1597, 1456, 1405, 1269 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.33 (1H, d, J=6.0 Hz), 5.40 (1H, dd, J=0.9, 6.0 Hz), 4.61
(1H, dddd, J=2.8, 11.6, 12.0 Hz), 4.09 (1H, ddd, J=2.8, 11.6, 12.0
Hz), 4.00 (1H, dddd, J=1.0, 4.2, 8.3, 15.5 Hz), 3.81 (1H, ddd,
J=1.4, 5.4, 11.6 Hz), 2.67 (1H, ddd, J=1.9, 3.2, 13.5 Hz), 2.63
(1H, dd, J=13.3, 16.8 Hz), 2.47 (1H, ddd, J=0.9, 4.0, 16.8 Hz),
2.39 (1H, dsept, J=1.6, 6.9 Hz), 2.02 (1H, ddd, J=5.6, 8.3, 14.0
Hz), 1.79 (1H, ddd, J=4.2, 6.9, 14.0 Hz), 1.20-1.75 (7H, m), 1.17
(1H, ddd, J=1.9, 3.8, 12.6 Hz), 0.89 (3H, d, J=6.7 Hz), 0.87 (3H,
d, J=6.9 Hz), 0.86 (3H, d, J=6.9 Hz), 0.70 (1H, t, J=13.5 Hz);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 192.6, 163.3, 107.1,
100.7, 76.4, 63.9, 58.8, 51.1, 41.6, 40.9, 37.3, 34.7, 31.4, 29.3,
24.2, 23.7, 22.2, 21.8, 18.8; LRMS (EI): 322 (61), 307 (18), 265
(27), 237 (56), 151 (21), 139 (33), 112 (17), 97 (100), 83 (26), 81
(34), 71 (17), 69 (60); HRMS Calcd for C.sub.19H.sub.30O.sub.4:
322.2144. Found: 322.2146; [.alpha.].sub.D.sup.20-57.8.degree. (c
1.5, CH.sub.2Cl.sub.2).
Formula 404
[0277] To a stirred solution of pyrone 403a (1.30 g, 4.04 mmol) in
40 mL of anhydrous MeOH was added CeCl.sub.3-7H.sub.2O (904 mg,
2.43 mmol) at rt. After stirring for 10 min, the mixture was cooled
to -40.degree. C. and NaBH.sub.4 (306 mg, 8.09 mmol) was added in
one portion. The mixture was stirred for an additional 15 min
before quenching with a 1:1 mixture of brine and H.sub.2O. The
aqueous layer was extracted with EtOAc (4.times.). The combined
organics were washed with sat. NaHCO.sub.3 and brine, dried over
Na.sub.2SO.sub.4 and concentrated to afford a clear oil.
Purification by flash chromatography (30% EtOAc/hexanes containing
1% Et.sub.3N) afforded unstable allylic alcohol 404 (1.08 g, 82%)
as a single diastereomer.
[0278] 404: IR (film) 3386, 2951, 2869, 1643, 1456, 1380, 1307,
1268, 1231 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
6.35 (1H, br d, J=5.4 Hz), 4.75 (1H, ddd, J=1.9, 1.9, 6.1 Hz),
4.36-4.46 (1H, m), 4.08-4.17 (1H, m), 4.08 (1H, ddd, J=2.7, 12.4,
12.4 Hz), 3.98 (1H, dddd, J=2.7, 5.2, 7.9, 10.9 Hz), 3.81 (1H, ddd,
J=1.5, 5.2, 11.5 Hz), 2.69 (1H, br d, J=13.5 Hz), 2.39 (1H, dsept,
J=1.7, 6.9 Hz), 2.18 (1H, dddd, J=1.7, 1.9, 6.4, 13.0 Hz), 1.90
(1H, ddd, J=6.6, 7.7, 14.0 Hz), 1.33-1.74 (9H, m), 1.17 (1H, ddd,
J=1.9, 4.4, 12.3 Hz), 0.90 (3H, d, J=6.2 Hz), 0.88 (6H, d, J=6.9
Hz), 0.69 (1H, t, J=12.7 Hz); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. 145.3, 105.5, 100.6, 71.4, 64.3, 63.0, 58.9, 51.2, 41.5,
37.7, 37.4, 34.9, 31.3, 29.2, 24.2, 23.7, 22.2, 21.8, 18.8;
[.alpha.].sub.D.sup.20+2.0.degree. (c 0.59, CHCl.sub.3).
Formula 405
[0279] To a solution of 1.08 g (3.32 mmol) of allylic alcohol 404
in 66 mL of ethylvinylether was added mercury(II)trifluoroacetate
(142 mg, 0.33 mmol) in a single portion at -10.degree. C. The
resulting colorless solution was stirred for 20 h at 5.degree. C.,
diluted with Et.sub.2O, washed with sat. NaHCO.sub.3 and brine,
dried over Na.sub.2SO.sub.4, and concentrated in vacuo to give a
clear, colorless oil. Rapid flash chromatography (10% EtOAc/hexanes
containing 1% Et.sub.3N) afforded 865 mg (74%) of the corresponding
allyl vinyl ether along with 208 mg (19%) of recovered 404. The
vinyl ether was immediately dissolved in 100 mL n-nonane and heated
at 145.degree. C. for 3.5 h. The solution was cooled to
70-80.degree. C. and the solvent was removed by short path
distillation at reduced pressure. The remaining residue was
purified by flash chromatography (10% EtOAc/hexanes) to provide
Claisen product 405 (612 mg, 71%) as a colorless oil.
[0280] 405: R.sub.f (30% EtOAc/hexanes)=0.75; IR (film) 2950, 2869,
1728, 1646, 1456, 1373, 1307, 1267 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 9.78 (1H, t, J=2.4 Hz), 5.89 (1H, dddd, J=2.0,
2.0, 4.7, 10.0 Hz), 5.64 (1H, dddd, J=1.3, 1.3, 2.5, 10.0 Hz),
4.57-4.66 (1H, m), 4.08 (1H, ddd, J=2.7, 12.1, 12.1 Hz), 3.90-4.01
(1H, m), 3.66-3.82 (2H, m), 2.69 (1H, ddd, J=1.9, 3.0, 13.4 Hz),
2.55 (2H, dd, J=2.4, 6.2 Hz), 2.40 (1H, dsept, J=1.6, 6.9 Hz),
1.12-2.14 (12H, m), 0.89 (3H, d, J=6.6 Hz), 0.88 (6H, d, J=7.1 Hz),
0.68 (1H, t, J=12.9 Hz); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
201.7, 128.5, 126.3, 100.5, 70.6, 70.5, 64.4, 59.1, 51.2, 48.5,
42.5, 37.4, 34.9, 31.5, 30.7, 29.1, 24.3, 23.7, 22.2, 21.9, 18.9;
MS (EI) 350 (51), 335 (45), 322 (14), 293 (65), 279 (15), 265 (52),
255 (15), 139 (17), 135 (29), 97 (28), 83 (43), 81 (100), 79 (17),
69 (27), 67 (24); HRMS Calcd for C.sub.21H.sub.34O.sub.4: 350.2457.
Found: 350.2466; [.alpha.].sub.D.sup.27 0.degree.
(CH.sub.2Cl.sub.2).
Formula 406
[0281] To a stirred mixture of trimethylsilyl
diethylphosphonoacetate (72 .mu.l, 76.7 mg, 0.286 mmol) in
anhydrous THF (1 mL) was added of n-butyllithium (2.5 M in hexanes,
109 .mu.L, 0.272 mmol) dropwise at -78.degree. C. After stirring
for 20 min at -78.degree. C. and 15 min at rt, the mixture was
cooled to -78.degree. C. and treated with a THF solution (1 mL) of
aldehyde 405 (45 mg, 0.129 mmol). The mixture was allowed to warm
to rt over 2 h and stirring was continued for 1 h. The mixture was
diluted with 50 mL EtOAc and acidified with 10 mL of a 0.1 N
aqueous NaHSO.sub.4 solution. The phases were separated and the
aqueous phase was extracted with EtOAc (3.times.). The combined
organic extracts were washed with brine, dried over MgSO.sub.4, and
concentrated to give a colorless oil. Rapid filtration through a
short pad of silica gel (20% acetone/benzene) gave a crude product
which was dissolved in 2 mL methanol. Palladium on activated
charcoal (10%, .about.5 mg) was added and the mixture was stirred
at room temperature for 18 h under balloon pressure of hydrogen
gas. Filtration through Celite.TM. and flash chromatography on
silica gel (40% EtOAc/hexanes containing 1% acetic acid) gave 21.8
mg (0.055 mmol, 43%) of ketal carboxylic acid 406 as a clear,
colorless oil. R.sub.f (40% EtOAc/hexanes+1% AcOH)=0.44; IR (film)
3500-2500, 2938, 2868, 1711, 1456, 1377, 1307, 1268, 1158, 1113
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 4.08 (ddd, 1H,
J=2.6, 12.4, 12.4 Hz), 3.98 (dddd, 1H, J=2.4, 6.6, 6.6, 10.7 Hz),
3.81 (ddd, 1H, J=1.2, 5.2, 11.5 Hz), 3.38-3.47 (m, 1H), 3.21-3.31
(m, 1H), 2.70 (br d, 1H, J=12.5 Hz), 2.29-2.47 (m, 3H), 1.62-1.86
(m, 5H), 1.34-1.62 (m, 11H), 1.07-1.30 (m, 3H), 0.78-0.98 (m, 1H),
0.88 (d, 6H, J=7.2 Hz), 0.88 (d, 3H, J=7.0 Hz), 0.67 (t, 1H, J=12.9
Hz); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 179.1, 100.5, 74.2,
64.6, 59.2, 51.2, 42.9, 37.3, 35.4, 34.9, 33.7, 31.50, 31.46, 31.3,
29.0, 24.2, 23.7, 23.6, 22.2, 21.8, 20.9, 18.8; HRMS Calcd for
C.sub.23H.sub.40O.sub.5: 396.2876. Found: 396.2870;
[.alpha.].sup.28.sub.D -10.5.degree. (c 1.66, CHCl.sub.3).
2B. Ketal Acid 408
Formula 407
[0282] To a solution of the aldehyde 405 (Example 2A, 274 mg, 0.778
mmol) in ethyl acetate (5 mL) was added Pd/C (10 mg) and the
atmosphere was exchanged to hydrogen, which was applied for 30 min.
The mixture was filtered and concentrated to give the corresponding
crude saturated aldehyde, which was directly used in the next
step.
[0283] A stock solution of Ipc.sub.2B(allyl) was prepared by first
dissolving (-)-Ipc.sub.2BOMe (700 mg, 2.22 mmol) in ether (4.15 mL)
at 0.degree. C. and adding 1M allyl magnesium bromide (1.78 mL,
1.78 mmol). The mixture was warmed to rt and stirred for 30 min. In
a separate flask, the aldehyde was dissolved in ether (4 mL) and
treated with the stock solution of Ipc.sub.2B(allyl) (0.3 M, 3.9
mL, 1.17 mmol) at -78.degree. C. After stirring for 2 h at
-78.degree. C., the mixture was treated with hydrogen peroxide
(30%, 2 mL) and sodium hydroxide (15%, 2 mL) and warmed to rt.
After another 2 h, the mixture was partitioned between ethyl
acetate and brine. The aqueous layer was extracted with ethyl
acetate. The combined organic layers were dried over sodium
sulfate, and concentrated to give the corresponding crude
homoallylic alcohol, which was directly used in the next step.
[0284] To a solution of the crude homoallylic alcohol in methylene
chloride (5 mL) at 0.degree. C. was added TBSOTf (534 .mu.L, 2.33
mmol) and diisopropyl ethyl amine (676 .mu.L, 3.89 mmol) and
stirred for 30 min. The mixture was directly loaded onto silica gel
and purified to give the corresponding alkene (237 mg, 60% in 3
steps).
[0285] To a solution of the alkene (10 mg, 0.0197 mmol) in
tert-butanol (1 mL) at rt was added a solution of KMnO.sub.4 (0.6
mg, 0.0039 mmol) and NaIO.sub.4 (17 mg, 0.0788 mmol) in water
(buffered pH 7, 1 mL). After 30 min, the reaction mixture was
quenched with sodium thiosulfate. The mixture was partitioned
between ethyl acetate and brine. The aqueous layer was extracted
with ethyl acetate. The combined organic layers were dried over
sodium sulfate, and concentrated. Column chromatography afforded
TBS ether 407 (6.3 mg, 71%).
[0286] 407: Rf=0.20 (25% ethyl acetate/hexane);
[.alpha.].sup.25.sub.D=22.6.degree. (c 0.58, CH.sub.2Cl.sub.2); IR
(neat)=2933, 1712, 1457, 1255, 1114 cm.sup.-1; .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 4.32 (m, 1H), 4.08 (m, 1H), 3.93 (m, 1H),
3.81 (m, 1H), 3.44 (m, 2H), 2.69 (d, J=13.2 Hz, 1H), 2.62 (dd,
J=15.2, 5.2 Hz, 1H), 2.48 (dd, J=15.2, 5.2 Hz, 1H), 2.40 (quint,
J=6.8 Hz, 1H), 1.85-1.15 (m, 20H), 0.88 (br s, 18H), 0.12 (s, 3H),
0.11 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 173.48,
100.44, 73.88, 73.52, 66.15, 64.26, 59.14, 51.24, 43.89, 43.25,
42.48, 37.43, 43.97, 32.13, 31.89, 30.93, 29.23, 25.74, 24.31,
23.77, 23.52, 22.25, 21.89, 19.15, 17.92, 4.77; HRMS: calcd for
(C.sub.29H.sub.54O.sub.6Si)=526.3689; found (M)=526.3687
Formula 408
[0287] To a solution of TBS ether 407 (9.3 mg, 0.0177 mmol) in THF
(0.5 mL) at rt was added pyridine (127 .mu.L) and HFpyridine (51
.mu.L, 1.77 mmol) and stirred for 18 h. The mixture was partitioned
between ethyl acetate and water (pH 4). The aqueous layer was
extracted with ethyl acetate. The combined organic extracts were
dried over sodium sulfate, concentrated in vacuo and the resulting
crude hydroxy acid was used in the next step without further
purification.
[0288] To a solution of crude hydroxy acid in methylene chloride
(1.5 mL) at rt was added TESCl (26 .mu.L, 0.0708 mmol) and TEA (19
.mu.L, 0.142 mmol) and the mixture was stirred for 4 h. The mixture
was treated with water (pH 4, 1 mL) and stirred for another 10 min.
The aqueous layer was extracted with ethyl acetate. The combined
organic extracts were dried over sodium sulfate and then
concentrated. The crude product was purified by silica gel
chromatography to give the TES acid 408 (7 mg, 75%-2 steps).
[0289] 408: R.sub.f=0.30 (33% ethyl acetate in hexane);
[.alpha.].sup.25.sub.D=34.0.degree. (c 0.42, CH.sub.2Cl.sub.2); IR
(film)=2951, 1711, 1457, 1378, 1114, 1005, 976 cm.sup.-1; .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 4.33 (m, 1H), 4.09 (m, 1H), 3.95
(m, 1H), 3.81 (m, 1H), 3.45 (m, 1H), 2.70 (d, J=14 Hz, 1H), 2.64
(dd, J=15.6, 5.2 Hz, 1H), 2.51 (dd, J=15.6, 5.2 Hz, 1H), 2.40 (m,
1H), 1.86-1.15 (m, 21H), 0.98 (t, J=8.0 Hz, 9H), 0.89 (m, 9H), 0.66
(q, J=8.0 Hz, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
173.06, 100.45, 73.94, 73.70, 66.33, 64.28, 59.12, 51.21, 43.90,
43.23, 42.48, 37.40, 34.96, 32.12, 31.86, 31.00, 29.20, 24.33,
23.77, 23.52, 22.23, 21.89, 19.00, 6.79, 4.73; HRMS: calcd for
(C.sub.29H.sub.54O.sub.6Si)=526.3689; found (M)=526.3717.
2C. 9-Hydroxy-9-t-Butyl L3 Linker Synthon 504
Formula 501
[0290] To a solution of aldehyde 402 (Example 2A supra, 803 mg,
3.16 mmol) in ether (10 mL) was added a solution of 4-pentenyl
magnesium bromide (0.8M in ether, 4.74 .mu.L, 3.79 mmol) at
-78.degree. C. and the mixture was stirred for 30 min. The reaction
was quenched with aq. sat. NH.sub.4Cl (10 mL) and allowed to warm
to rt. The mixture was then extracted with EtOAc (3.times.10 mL)
and the combined organics were dried over MgSO.sub.4, and
concentrated in vacuo. The resulting residue was dissolved in
CH.sub.2Cl.sub.2 (15 mL) and Dess-Martin Periodinane (2.02 g, 4.74
mmol) was added at rt. After 3 hours, sat. aq. Na.sub.2SO.sub.3 (10
mL) and sat. aq. NaHCO.sub.3 (10 mL) was added. The mixture was
extracted with CHCl.sub.3 (3.times.10 mL), washed with sat. aq.
NaHCO.sub.3 (10 mL), dried over MgSO.sub.4, and concentrated in
vacuo. The residue was purified by column chromatography (90%
EtOAc/hexane) to obtain ketone 501 (730 mg, 71.6%) as a colorless
oil. 501: IR (film) 2957, 2362, 1716, 1637 cm.sup.-1; .sup.1H NMR
(300 MHz, CDCl.sub.3) 6, 0.62-0.92 (11H, m), 1.12-1.70 (9H, m) 2.03
(2H, dd, J=14.77, 7.63 Hz), 2.24-2.71 (6H, m), 3.79 (1H, ddd,
J=11.53, 5.22, 1.37 Hz), 4.10 (1H, td, J=11.88, 2.81 Hz), 4.22-4.31
(1H, m), 4.94-5.03 (2H, m), 5.68 (1H, m); .sup.13C NMR (75 MHz,
CDCl.sub.3) 6, 18.82, 21.70, 22.13, 22.37, 23.62, 24.18, 28.59,
31.34, 32.95, 34.76, 37.32, 43.73, 49.33, 51.09, 58.81, 65.39,
100.73, 115.21, 138.04, 209.56; HRMS (EI) Calc'd. for
C.sub.20H.sub.34O.sub.3: 322.2508. Found: 322.2497.
[.alpha.].sub.D.sup.25=5.52.degree. (c 4.79, CH.sub.2Cl.sub.2).
502.1 (Formula 502 where R.sup.8 is t-butyl)
[0291] To a solution of ketone 501 (35 mg, 0.109 mmol) in ether
(0.6 mL) was added t-BuLi (1.6 M in pentane, 75 .mu.L, 0.12 mmol)
dropwise at -78.degree. C. The mixture was stirred at rt for 30
min. and then quenched with sat. aq. NH.sub.4Cl (2 mL). The mixture
was allowed to warm to rt and was then extracted with EtOAc
(3.times.5 mL). The combined organics were washed with brine, dried
over Na.sub.2SO.sub.4, and concentrated in vacuo. Chromatography
(10% EtOAc/hexane) afforded a major diastereomer A31a (17.5 mg,
42%) along with a minor diastereomer A31b. (7.4 mg, 18%) as
colorless oils (structures not shown). A31a: IR (film): 2956, 1639,
1456 cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3) 6, 0.73 (1H, t,
J=12.88 Hz), 0.89-0.94 (18H, m), 1.17-1.76 (14H, m), 2.00-2.09 (2H,
m), 2.38-2.42 (1H, m), 2.73 (1H, d, J=13.73 Hz), 3.47 (1H, s), 3.79
(1H, dd, J=11.60, 5.06 Hz), 4.12 (1H, td, J=11.97, 2.44 Hz),
4.25-4.29 (1H, m), 4.91 (1H, d, J=10.17 Hz), 4.98 (1H, d, J=17.63
Hz), 5.77-5.84 (1H, m); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
19.07, 22.20, 22.25, 23.15, 23.45, 24.66, 25.43, 25.52, 28.98,
31.80, 34.36, 34.51, 34.60, 36.84, 37.54, 39.29, 39.63, 51.30,
58.92, 67.73, 79.0, 101.26, 114.31, 139.03; HRMS (EI) Calc'd. for
C.sub.24H.sub.44O.sub.3: 380.3290. Found: 380.3281.
[.alpha.].sub.D.sup.25=5.89.degree. (c 0.85, CDCl.sub.3).
[0292] To a solution of major diastereomer A31a (332 mg, 0.872
mmol) in THF (5 mL) was added KHMDS (0.5M in toluene, 5.24 mL, 2.62
mmol) in an ice bath. TMSCl (284 mg, 2.62 mmol) was added and the
mixture was stirred for 30 minutes. The mixture was quenched with
sat. aq. NH.sub.4Cl (5 mL) and extracted with EtOAc (3.times.5 mL).
The combined organics were washed with brine (5 mL), dried over
MgSO.sub.4, and then concentrated in vacuo. Chromatography (5%
EtOAc/hexane) providing 502.1 (395 mg, 100%) as a white solid.
502.1: m.p.=60.0.degree. C.; IR (film) 2953, 1642, 1455 cm.sup.-1;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta., 0.132 (6H, s), 0.134
(3H, s), 0.67-1.71 (33H, m), 1.95-1.99 (2H, m), 2.36-2.46 (1H, m),
2.71 (1H, app d, J=14.3 Hz), 3.74-3.79 (1H, m), 3.88-3.94 (1H, m),
4.07 (1H, td, J=11.75, 2.96 Hz), 4.95 (1H, d, J=10.41 Hz), 5.00
(1H, d, J=17.33 Hz), 5.74-5.85 (1H, m); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 2.9, 5.3, 18.9, 21.7, 22.2, 23.8, 24.3, 25.3,
26.2, 29.1, 34.2, 34.8, 35.0, 37.6, 39.2, 40.0, 51.3, 59.3, 65.6,
82.9, 100.7, 114.6, 138.7; HRMS (EI) Calc'd. for
C.sub.27H.sub.52O.sub.3Si: 452.3686. Found: 452.3670;
[.alpha.].sub.D.sup.26=-11.33.degree. (c 1.56, CDCl.sub.3).
503.1 (Formula 503 where R.sup.8 is t-butyl)
[0293] To a solution of 502.1 (98 mg, 0.216 mmol) in
CH.sub.2Cl.sub.2 (8 mL) and MeOH (0.5 mL) was bubbled O.sub.3 at
-78.degree. C. until a blue color persists. Nitrogen gas was then
used to purge the system and (EtO).sub.3P (54 mg, 0.324 mmol) was
subsequently added. The mixture was stirred for 3 h and then slowly
warmed to rt. Sat. aq. Na.sub.2SO.sub.3 (10 mL) was added and the
mixture was extracted with CHCl.sub.3 (3.times.10 mL). The combined
organics were then washed with brine (10 mL), dried over MgSO.sub.4
and concentrated in vacuo. Chromatography (0.5% EtOAc/hexane)
afforded 503.1 (63 mg, 64.1%) as a colorless oil. 503.1: IR (film)
2941, 1715, 1454 cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 0.134 (9H, s), 0.73 (1H, t, J=13.13 Hz), 0.9-1.81 (32H, m),
2.34-2.42 (3H, m), 2.72 (1H, d, J=12.29 Hz), 3.77 (1H, dd, J=11.59,
4.58 Hz), 3.92-3.96 (1H, m), 4.08 (1H, td, J=12.28, 2.01 Hz), 9.77
(1H, s); .sup.13C NMR (100 MHz, CDCl.sub.3) 6, 3.39, 18.98, 19.29,
22.60, 24.23, 24.71, 26.75, 29.76, 34.41, 35.15, 37.77, 38.01,
39.66, 42.95, 45.26, 51.67, 59.64, 65.90, 83.08, 100.99, 202.26;
HRMS (EI) Calc'd. for C.sub.26H.sub.50O.sub.4Si: 454.3478. Found:
452.3475; [.alpha.].sub.D.sup.27=-6.33.degree. (c 2.9,
CH.sub.2Cl.sub.2). ##STR56##
504.1 (Formula 504 where R.sup.8 is t-butyl)
[0294] To (-)-Ipc.sub.2BOMe (108.8 mg, 0.34 mmol) (weighed in an
inert atmosphere) was added diethyl ether (340 .mu.L). The flask
was cooled to -78.degree. C. and allylmagnesium bromide (1.0 M, 310
.mu.L, 0.31 mmol) was added. The precipitous mixture was stirred
for 15 min. at -78.degree. C. and then slowly warmed to rt and
stirred for 1 hour. Diethyl ether (340 mL) was added and then a
pre-cooled solution of aldehyde 503.1 (40.1 mg, 0.109 mmol) in
diethyl ether (160 .mu.L) was added dropwise. The suspension was
stirred for 1 h, then the temperature was slowly raised to rt and
3N NaOH (240 .mu.L), 30% hydrogen peroxide (100 .mu.L) and diethyl
ether (400 .mu.L) was added. The biphasic mixture was refluxed for
1 h and then quenched with water (9 mL) and extracted with ethyl
acetate (4.times.10 mL). The combined organics were washed with
brine (10 mL), dried over sodium sulfate and the solvent was
removed in vacuo. Chromatography on silica gel (12.5% EtOAc/hexane)
afforded the crude homoallylic alcohol as a yellow oil. To this oil
in methylene chloride (500 .mu.L) was added imidazole (19 mg, 0.276
mmol) and TBSCl (21 mg, 0.138 mmol). The reaction was sealed and
stirred for 14 h. The reaction was then quenched with a saturated
solution of sodium bicarbonate (2 mL) and extracted with ethyl
acetate (3.times.5 mL). The combined organics were washed with
brine (5 mL), dried over sodium sulfate, filtered and the solvent
was removed in vacuo. Filtration over silica gel (5% EtOAc/hexane)
afforded a crude silyl ether A34 as a yellow oil (structure not
shown). A34: IR (film) 2956, 1644, 1462 cm.sup.-1; .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 0.05 (6H, s), 0.05 (9H, s), 0.7-1.70 (42H,
m), 2.21 (2H, d, J=6.48 Hz), 2.38-2.44 (2H, m), 2.72 (2H, d,
J=13.36 Hz), 3.71 (1H, t, J=5.43 Hz), 3.77 (1H, dd, J=11.53, 3.59
Hz), 3.88-3.93 (1H, m), 4.07 (1H, td, J=1191, 3.06 Hz), 5.02 (1H,
s), 5.06 (1H, d, J=3.66 Hz), 5.77-5.88 (1H, m); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. -4.06, 3.39, 19.38, 21.48, 22.23, 22.72,
24.25, 24.78, 26.27, 26.69, 29.52, 34.58, 35.25, 38.06, 38.38,
38.50, 39.68, 42.02, 43.11, 59.68, 51.72, 65.98, 72.08, 83.20,
100.98, 117.06, 135.66; [.alpha.].sub.D=-4.18.degree. (c 1.0,
CDCl.sub.3).
[0295] To crude silyl ether A34 in t-butanol (2.3 mL), water (1.4
mL) and pH 7 phosphate buffer (465 .mu.L) was added NaIO.sub.4 (79
mg, 0.368 mmol) followed by KMnO.sub.4 (2.9 mg, 0.0184 mmol). The
purple solution was stirred for 3 h and then water (3 mL) was
added. This mixture was then extracted with ethyl acetate
(4.times.5 mL) and the combined organics were washed with brine (5
mL), dried over sodium sulfate and filtered. The solvent was
removed in vacuo and chromatography on silica gel (12.5% EtOAc,
0.1% acetic acid/hexanes) afforded acid 504.1 as well as the
.beta.-C3 diastereomeric alcohol. 504.1: IR (film): 3433, 2956,
1712, 1461 cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
0.08 (3H, s), 0.09 (3H, s), 0.13 (6H, s), 0.70-1.70 (48H, m),
2.35-2.55 (3H, m), 2.72 (1H, d, J=12.74 Hz), 3.78 (1H, dd, J=11.34,
3.97 Hz), 3.90-3.92 (1H, m), 4.75 (1H, dd, J=12.45, 11.97 Hz), 4.15
(1H, t, J=4.89 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
-4.53, -4.10, 3.39, 18.32, 19.39, 21.29, 22.29, 22.66, 24.23,
24.75, 26.12, 26, 72, 29.64, 34.49, 35.19, 38.04, 38.38, 38.81,
39.71, 42.11, 43.08, 51.67, 59, 65, 65.93, 69.51, 83.10, 101.01,
176.69; [.alpha.].sub.D.sup.28=-8.19.degree. (c 2.70,
CDCl.sub.3).
2D. 9-t-Butyl L4 Linker Synthon 508
[0296] ##STR57##
508.1 (Formula 508 where R.sup.8 is t-butyl)
[0297] To (-)-Ipc.sub.2BOMe (108.8 mg, 0.34 mmol) (weighed in an
inert atmosphere) was added diethyl ether (340 .mu.L). The flask
was cooled to -78.degree. C. and allylmagnesium bromide (1.0 M, 310
.mu.L, 0.31 mmol) was added. The precipitous mixture was stirred
for 15 min. at -78.degree. C. and then slowly warmed to rt and
stirred for 1 hour. Diethyl ether (340 mL) was added and then a
pre-cooled solution of an aldehyde 506 (0.109 mmol) (structure not
shown) prepared as described for compound 10 in Wender et al.
(1998c) in diethyl ether (160 .mu.L) was added dropwise. The
suspension was stirred for 1 h, then the temperature was slowly
raised to rt and 3N NaOH (240 .mu.L), 30% hydrogen peroxide (100
.mu.L) and diethyl ether (400 .mu.L) was added. The biphasic
mixture was refluxed for 1 h and then quenched with water (9 mL)
and extracted with ethyl acetate (4.times.10 mL). The combined
organics were washed with brine (10 mL), dried over sodium sulfate
and the solvent was removed in vacuo. Chromatography on silica gel
(12.5% EtOAc/hexane) afforded the crude homoallylic alcohol as a
yellow oil. To this oil in methylene chloride (500 .mu.L) was added
imidazole (19 mg, 0.276 mmol) and TBSCl (21 mg, 0.138 mmol). The
reaction was sealed and stirred for 14 h. The reaction was then
quenched with a saturated solution of sodium bicarbonate (2 mL) and
extracted with ethyl acetate (3.times.5 mL). The combined organics
were washed with brine (5 mL), dried over sodium sulfate, filtered
and the solvent was removed in vacuo. Filtration over silica gel
(5% EtOAc/hexane) afforded a crude silyl ether as a yellow oil. To
this oil in t-butanol (2.3 mL), water (1.4 mL) and pH7 phosphate
buffer (465 .mu.L) was added NaIO.sub.4 (79 mg, 0.368 mmol)
followed by KMnO.sub.4 (2.9 mg, 0.0184 mmol). The purple solution
was stirred for 3 h and then water (3 mL) was added. This mixture
was then extracted with ethyl acetate (4.times.5 mL) and the
combined organics were washed with brine (5 mL), dried over sodium
sulfate and filtered. The solvent was removed in vacuo and
chromatography on silica gel (12.5% EtOAc, 0.1% acetic
acid/hexanes) afforded acid 508.1 (34 mg, 56%) as a colorless oil.
508.1: R.sub.f(15% ethyl acetate/hexanes)=0.17; IR (film)
2700-3300, 2954, 2869, 1713, 1107, 837, 776 cm.sup.-1; 1H NMR (300
MHz, CDCl.sub.3) .delta. 0.08 (6H, s), 0.69 (1H, t, J=13.1 Hz),
0.76-0.90 (27H, m), 1.17-1.25 (3H, m), 1.39-1.61 (6H, m), 1.70-1.82
(3H, m), 2.40 (1H, dsept, J=7.2, 0.9 Hz), 2.58 (1H, dd, J=13.1, 5.6
Hz), 2.68 (1H, br d, J=12.6 Hz), 2.91 (1H, t, J=4.8 Hz), 3.41 (1H,
dd, J=5.9, 2.3 Hz), 3.63-3.77 (2H, m), 3.82 (1H, dd, J=11.7, 4.2
Hz), 4.066 (1H, td, J=12.0, 2.7 Hz), 4.23-4.29 (1H, m), 9.20-9.40
(1H, br s); .sup.13C NMR (100 MHz, C.sub.6D.sub.6) .delta. -5.1,
-4.8, 17.8, 18.9, 21.9, 22.1, 23.7, 24.3, 25.6, 26.0, 29.1, 31.8,
34.9, 35.7, 37.3, 38.7, 41.8, 51.3, 59.2, 66.6, 66.9, 67.2, 84.5,
174.7; HRMS (FAB) Calc'd. for C.sub.30H.sub.58O.sub.6Si: 542.4002,
Found: 542.4005; [.alpha.].sub.D.sup.22=-5.88.degree. (c 1.67,
CH.sub.2Cl.sub.2).
2E. Linker Synthon 507
[0298] To (-)-Ipc.sub.2BOMe (108.8 mg, 0.34 mmol) (weighed in an
inert atmosphere) was added diethyl ether (340 .mu.L). The flask
was cooled to -78.degree. C. and allylmagnesium bromide (1.0 M, 310
.mu.L, 0.31 mmol) was added. The precipitous mixture was stirred
for 15 min. at -78.degree. C. and then slowly warmed to rt and
stirred for 1 hour. Diethyl ether (340 mL) was added and then a
pre-cooled solution of an aldehyde 506 (0.109 mmol) (structure not
shown) prepared as described for compound 9 in Wender et al.
(1998c) in diethyl ether (160 .mu.L) was added dropwise. The
suspension was stirred for 1 h, then the temperature was slowly
raised to rt and 3N NaOH (240 .mu.L), 30% hydrogen peroxide (100
.mu.L) and diethyl ether (400 .mu.L) was added. The biphasic
mixture was refluxed for 1 h and then quenched with water (9 mL)
and extracted with ethyl acetate (4.times.10 mL). The combined
organics were washed with brine (10 mL), dried over sodium sulfate
and the solvent was removed in vacuo. Chromatography on silica gel
(12.5% EtOAc/hexane) afforded the crude homoallylic alcohol A40
(structure not shown) as a yellow oil. Rf (35% ethyl
acetate/hexanes)=0.65; IR (film) 3455, 2950, 2868, 1456, 1373,
1308, 1265, 1110, 997 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 0.68 (1H, t, J=13.5 Hz), 0.83-0.92 (10H, m), 1.39-1.52 (5H,
m), 1.57-1.72 (3H, m), 1.71 (1H, d, J=4.9 Hz), 2.24 (1H, t, J=4.9
Hz), 2.35-2.42 (2H, m), 2.70 (3H, br d, J=12.4 Hz), 3.46-3.60 (3H,
m), 3.62-3.70 (1H, m), 3.77-3.95 (3H, m), 4.11 (1H, dd, J=11.9, 2.7
Hz), 5.07 (2H, dd, J=9.5, 1.9 Hz), 5.70-5.91 (1H, m); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. 13.9, 16.4, 17.5, 21.1, 22.0, 23.1,
29.1, 30.7, 31.1, 31.8, 33.7, 37.3, 46.1, 51.1, 56.9, 64.7, 65.0,
66.8, 113.3, 129.3; [.alpha.].sub.D.sup.20=2.5.degree. (c 0.8,
CDCl.sub.3).
[0299] To A40 in methylene chloride (500 .mu.L) was added imidazole
(19 mg, 0.276 mmol) and TBSCl (21 mg, 0.138 mmol). The reaction was
sealed and stirred for 14 h. The reaction was then quenched with a
saturated solution of sodium bicarbonate (2 mL) and extracted with
ethyl acetate (3.times.5 mL). The combined organics were washed
with brine (5 mL), dried over sodium sulfate, filtered and the
solvent was removed in vacuo. Filtration over silica gel (5%
EtOAc/hexane) afforded a crude silyl ether A41 (structure not
shown) as a yellow oil. Rf (35% ethyl acetate/hexanes)=0.65; IR
(film): 2953, 2864, 1641, 1472, 1372, 1255, 1108, 830 cm.sup.-1;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.05 (3H, s), 0.05 (3H,
s), 0.67 (1H, t, J=13.5 Hz), 0.81-0.96 (18H, m), 1.11-1.20 (1H, m),
1.35-1.47 (4H, m), 1.52-1.71 (5H, m), 2.22 (1H, br s), 2.35-2.42
(2H, m), 2.70 (3H, br d, J=12.2 Hz), 3.38-3.54 (4H, m), 3.78-3.97
(3H, m), 4.10 (1H, dd, J=11.9, 2.8 Hz), 5.05 (2H, dd, J=9.4, 1.8
Hz), 5.69-5.91 (1H, m); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
-9.5, -9.1, 13.3, 14.1, 17.1, 17.5, 18.9, 19.5, 21.1, 24.1, 27.0,
30.2, 31.8, 32.0, 32.6, 37.5, 46.4, 54.4, 59.5, 61.9, 62.8, 64.1,
95.6, 112.0, 130.0; [.alpha.].sub.D.sup.20=16.2.degree. (c 1.0,
CH.sub.2Cl.sub.2).
[0300] To A41 in t-butanol (2.3 mL), water (1.4 mL) and pH 7
phosphate buffer (465 .mu.L) was added NaIO.sub.4 (79 mg, 0.368
mmol) followed by KMnO.sub.4 (2.9 mg, 0.0184 mmol). The purple
solution was stirred for 3 h and then water (3 mL) was added. This
mixture was then extracted with ethyl acetate (4.times.5 mL) and
the combined organics were washed with brine (5 mL), dried over
sodium sulfate and filtered. The solvent was removed in vacuo and
chromatography on silica gel (12.5% EtOAc, 0.1% acetic
acid/hexanes) afforded acid 507. IR (film): 2700-3300, 2952, 2866,
1738, 1471, 1373, 1307, 1146, 1103, 837, cm.sup.-1; .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 0.06 (3H, s), 0.07 (3H, s), 0.67 (1H,
app t, J=13.1 Hz), 0.81-0.89 (19H, m), 1.14-1.20 (1H, m), 1.35-1.47
(5H, m), 1.50-1.82 (4H, m), 2.34-2.41 (1H, m), 2.45-2.53 (2H, m),
2.69 (3H, br d, J=13.7 Hz), 3.44-3.51 (4H, m), 3.80 (1H, dd,
J=11.5, 3.9 Hz), 3.87-3.95 (1H, m), 4.02-4.09 (1H, m), 4.22-4.29
(1H, m); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. -9.6, 13.1,
14.1, 17.1, 17.5, 18.9, 19.5, 20.9, 24.2, 26.9, 30.1, 32.0, 32.3,
32.6, 37.7, 46.4, 54.3, 59.6, 61.9, 62.1, 95.7, 171.5; HRMS (FAB)
Calc'd. for C.sub.26H.sub.50O.sub.6Si: 486.3379, Found: 486.3377;
[.alpha.].sub.D.sup.20=9.4.degree. (c 1.3, CH.sub.2Cl.sub.2).
2F. Ether Diester Linker Synthon 606
[0301] The following procedure, referred to as the "general
isolation procedure", was used to purify various reaction products
below. The reaction mixture is quenched by dropwise addition of
saturated aqueous ammonium chloride, and the resultant mixture is
allowed to partition between solvent and brine or water. The
aqueous layer is extracted with 1 to 3 portions of ethyl acetate.
The combined organic extracts are dried over sodium sulfate and
concentrated in vacuo.
Formula 602
[0302] To a solution of 3-(p-methoxybenzyloxy)propyl allyl ether
601 (1.0 g, 4.3 mmol) in THF (10 mL) was added 9-BBN (20.6 mL of
0.5 M solution in THF, 10.3 mmol), and the mixture was stirred for
2 h at rt. Hydrogen peroxide (30%, 10 mL) and sodium hydroxide
(15%, 10 mL) were added and the mixture was stirred for 3 h. The
general isolation procedure afforded crude product which was
purified further by silica gel chromatography to give the expected
purified alcohol A51 (870 mg, 75%) (structure not shown).
R.sub.f=0.25 (50% ethyl acetate in hexane); IR (neat)=3423, 2934,
2864, 1612, 1513, 1248, 1098 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 7.25 (d, J=8.7 Hz, 2H), 6.87 (d, J=8.7 Hz, 2H),
4.43 (s, 2H), 3.80 (s, 3H), 3.75 (q, J=5.7 Hz, 2H), 3.60 (t, J=5.7
Hz, 2H), 3.52 (m, 4H), 2.46 (br t, 1H), 1.84 (m, 4H); .sup.13C NMR
(75 MHz, CDCl.sub.3) .delta. 129.36, 113.83, 72.63, 70.34, 68.33,
66.89, 62.20, 55.23, 31.88, 29.96.
[0303] To a solution of DMSO (1.38 mL, 19.5 mmol) in methylene
chloride (5 mL) at -78.degree. C. was added oxalyl chloride (850:L,
9.74 mmol) dropwise. After 5 min, alcohol A51 from the preceding
step (870 mg, 6.49 mmol) in methylene chloride (2 mL) was added and
the mixture was stirred for another 20 min. TEA (triethylamine,
4.31 mL, 32.5 mmol) was added. After 10 min, the mixture was warmed
to rt. The standard isolation procedure afforded the expected
aldehyde 602 (760 mg, 89%). R.sub.f=0.40 (33% ethyl acetate in
hexane); IR (neat)=2863, 1724, 1612, 1513, 1248, 1098 cm.sup.-1;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 9.75 (s, 1H), 7.25 (d,
J=9.0 Hz, 2H), 6.87 (d, J=9.0 Hz, 2H), 4.42 (s, 2H), 3.80 (s, 3H),
3.74 (t, J=6.0 Hz, 2H), 3.51 (m, 4H), 2.62 (m, 2H), 1.84 (quint,
J=6.3 Hz, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 201.49,
159.26, 130.65, 129.31, 113.78, 72.57, 68.08, 66.69, 64.40, 55.19,
43.75, 29.86.
Formula 603
[0304] (-)-Ipc.sub.2BOMe (1.91 g, 6.04 mmol) in ether (4 mL) was
treated with allylmagnesium bromide (4.83 mL, 4.83 mmol) at rt.
After 30 min, the mixture was cooled to -78.degree. C. The aldehyde
602 (370 mg, 2.76 mmol) in ether (1.5 mL) was added and stirred for
2 h at -78.degree. C. 15% NaOH (1.5 mL) and 30% hydrogen peroxide
(1.5 mL) were added and the mixture was warmed to rt. After 2 h,
the mixture was diluted with ethyl acetate and washed with brine.
Column chromatography yielded the expected alcohol A53 (383 mg,
80%) (structure not shown). Alcohol A53 (150 mg, 0.852 mmol) in
methylene chloride (4 mL) was treated with TBSCl (168 mg, 1.11
mmol) and imidazole (116 mg, 1.7 mmol) at rt. After overnight, the
reaction was worked up by the standard procedure. Column
chromatography afforded expected TBS alkene ether 603 (195 mg,
79%). 603: R.sub.f=0.50 (10% ethyl acetate in hexane);
[.alpha.].sup.25.sub.D=13.6.degree. (c 1.08, CH.sub.2Cl.sub.2); IR
(neat)=2952, 2856, 1717, 1613, 1513, 1471, 1362, 1248, 1110
cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.26 (d, J=8.4
Hz, 2H), 6.88 (d, J=8.4 Hz, 2H), 5.81 (m, 1H), 5.04 (m, 2H), 4.43
(s, 2H), 3.86 (s, 3H), 3.49 (m, 7H), 2.23 (m, 2H), 1.87 (m, 2H),
1.69 (m, 2H), 0.89 (s, 9H), 0.06 (s, 3H), 0.05 (s, 3H); .sup.13C
NMR (100 MHz, CDCl.sub.3) .delta. 159.09, 134.96, 130.60, 116.93,
113.71, 72.59, 68.91, 67.80, 67.48, 67.18, 55.23, 42.28, 36.62,
30.16, 25.85, 18.08, -4.39, -4.76
Formula 605
[0305] Alkene ether 603 (195 mg, 0.672 mmol) in tert-butanol-water
(pH 7) (1:1, 4 mL) was treated with KMnO.sub.4 (11 mg, 0.0672 mmol)
and sodium periodate (548 mg, 2.56 mmol) in water (0.5 mL). After
30 min, the reaction was quenched with sodium thiosulfate. The
mixture was diluted with ethyl acetate. The organic layer was
washed with brine, dried over sodium sulfate, and concentrated to
give a crude acid 604. The crude acid in methylene chloride (3 mL)
was treated with SEMCl (2-(trimethylsilyl)ethoxymethyl chloride,
301:L, 1.7 mmol) and TEA (452:L, 3.4 mmol) at rt and stirred for 2
h. The reaction was worked up by the standard procedure to afford
the expected SEM ester 605.
[0306] SEM ester 605 in wet methylene chloride (3 mL) was treated
with DDQ (291 mg, 1.28 mmol). After 1 h, the mixture was directly
purified by silica gel chromatography to give the alcohol product
606 (86 mg, 29% for 3 steps). 606: R.sub.f=0.25 (25% ethyl acetate
in hexane); [.alpha.].sup.25.sub.D=0.4.degree. (c 0.82,
CH.sub.2Cl.sub.2); IR (neat)=3458, 2955, 1741, 1472, 1378, 1250,
1116 cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.25 (q,
J=2.8 Hz, 2H), 4.25 (quint, J=6.4 Hz, 2H), 3.71 (m, 4H), 3.52 (m,
4H), 2.50 (d, J=6.0 Hz, 2H), 2.44 (br, 1H), 1.79 (m, 4H), 0.94 (t,
J=8.4 Hz, 2H), 0.85 (s, 9H), 0.05 (s, 3H), 0.03 (s, 3H), 0.00 (s,
9H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 171.04, 88.97,
69.88, 67.88, 67.25, 66.45, 61.81, 42.84, 37.12, 32.02, 25.71,
17.97, 17.91, -1.48, -4.81, -4.86
2G. C5 Ester-Open C7 Linker Synthon 513
[0307] ##STR58##
[0308] To menthone aldehyde 402 (400 mg, 1.56 mmol) from Example 2A
in DMF (12 mL) was added prenyl bromide (396 mg, 1.56 mmol)
followed by indium powder (359 mg, 3.13 mmol) at rt. After 30 min.,
the reaction was diluted with EtOAc (20 mL) and saturated aqueous
ammonium chloride (15 mL). The layers were separated and the
aqueous layer was re-extracted with EtOAc (3.times.10 mL). The
combined organics were washed with brine (15 mL) and dried over
sodium sulfate. The solvent was removed in vacuo and chromatography
(7.5% EtOAc/pentane) to afford 509a (105 mg, 21%) and 509b (321 mg,
63%).
[0309] Undesired isomer 509a can be recycled by the following
procedure: To 509a (320.8 mg, 0.98 mmol) in methylene chloride (3
mL) was added Dess-Martin Periodinane (707 mg, 1.67 mmol) and
stirred for 1 h at room temperature. The reaction was diluted with
methylene chloride (3 mL), saturated sodium bicarbonate (3 mL) and
sodium thiosulfate (3 mL) and stirred for 1 h. The layers were
separated and the aqueous layer was re-extracted with EtOAc
(4.times.5 mL). The combined organics were dried over sodium
sulfate and the solvent was removed in vacuo. The crude ketone was
dissolved in MeOH (17.3 mL) and CeCl.sub.3.7H.sub.2O (1.83 g, 4.9
mmol) was added and stirred for 5 min. The solution was cooled to
-50.degree. C. and NaBH.sub.4 (74.5 mg, 19.6 mmol) was added. The
reaction was stirred for 30 min. and then poured into a separatory
funnel containing EtOAc (30 mL), water (18 mL) and brine (30 mL).
The layers were separated and the aqueous layer was extracted with
EtOAc (3.times.10 mL). The combined organics were dried over sodium
sulfate and the solvent was removed in vacuo. Chromatography
(7.5%-20% EtOAc/pentane) provided 509b (177 mg, 53%) and 509a (56
mg, 18%). 509a: R.sub.f (7.5% EtOAc/pentane)=0.186; IR (film):
3496.8, 2953.9, 2869.6, 1458.0, 1374.4, 1308.3, 1266.5, 1157.8,
1130.7, 1102.0, 977.6, 912.2 cm.sup.-1; .sup.1H-NMR (300 MHz,
CDCl.sub.3) .delta. 0.68 (1H, t, J=12.9 Hz), 0.85-0.89 (9H, m),
0.98 (3H, s), 0.99 (3H, s), 1.14-1.76 (10H, m), 2.38 (1H, sept,
J=6.9 Hz), 2.73 (1H, br. d, J=13.5 Hz), 3.59 (1H, br. d, J=9.9 Hz),
3.78 (1H, dd, J=11.7, 5.4 Hz), 4.02 (1H, d, J=11.4), 4.11 (1H, d,
J=13.2), 5.00-5.09 (2H, m), 5.81 (1H, dd, J=17.6, 11.0 Hz);
.sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. 18.8, 21.7, 22.0, 22.4,
23.1, 23.8, 24.3, 29.1, 31.9, 34.9, 37.4, 38.5, 41.2, 51.2, 59.1,
64.5, 73.5, 100.4, 113.2, 145.4; HRMS calc'd for
C.sub.20H.sub.36O.sub.3: 324.2664; found: 324.2664;
[.alpha.].sup.24.sub.d:+19.36.degree. (c 8.16, CH.sub.2Cl.sub.2).
509b: R.sub.f (7.5% EtOAc/pentane)=0.37; IR (film): 3520.4, 2953.6,
2870.2, 1455.8, 1375.6, 1309.4, 1266.0, 1130.3, 1090.0, 973.3,
914.4 cm.sup.-1; .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 0.73
(1H, t, J=13 Hz), 0.83-0.92 (9H, m), 1.00 (3H, s), 1.01 (3H, s),
1.18-1.77 (10H, m), 2.39 (1H, sept, J=7 Hz), 2.73 (1H, br. d,
J=13.5 Hz), 3.53 (1H, d, J=9.9 Hz), 3.57 (1H, s), 3.79 (1H, dd,
J=11.7, 4.8 Hz), 4.00-4.15 (2H, m), 4.96-5.02 (2H, m), 5.87 (1H,
dd, J=17.1, 1.1 Hz); .sup.13C-NMR (75 MHz, CDCl.sub.3) .delta.
18.9, 21.9, 22.2, 22.3, 23.3, 23.8, 24.2, 28.9, 31.8, 34.6, 37.3,
38.0, 41.1, 50.9, 58.8, 70.3, 78.9, 101.0, 111.9, 145.7; HRMS
calc'd for C.sub.20H.sub.36O.sub.3: 324.2664; found: 324.2665;
[.alpha.].sup.25.1.sub.d:-1.94.degree. (c 9.75,
CH.sub.2Cl.sub.2).
Formula 510
[0310] To a solution of 509b (282 mg, 0.87 mmol) in MeOH (3.3 mL)
and methylene chloride (13 mL) at -78.degree. C. was bubbled ozone
for 5 min. or until a blue color persisted. The solution was then
purged with nitrogen for 5 min. NaBH.sub.4 (138 mg, 3.65 mmol) was
then added and stirred for 1 h at -78.degree. C., and 2 h at
0.degree. C. Saturated ammonium chloride (15 mL) and water (5 mL)
were then poured into the reaction and the layers were separated.
The aqueous layer was extracted with methylene chloride (4.times.5
mL). The combined organics were dried over sodium sulfate and the
solvent was removed in vacuo, yielding crude diol 510: R.sub.f (20%
EtOAc/pentane)=0.195; IR (film): 3452.3, 2954.1, 2871.3, 1455.5,
1383.4, 1308.9, 1266.9, 1131.4, 1047.2, 972.6 cm.sup.-1;
.sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 0.74 (1H, t, J=13 Hz),
0.85-0.89 (12H, m), 0.92 (3H, d, J=6.6 Hz), 1.12-1.25 (2H, m),
1.39-1.78 (7H, m), 2.39 (1H, sept, J=6.9 Hz), 2.73 (1H, br. d,
J=13.5 Hz), 3.48 (3H, m), 3.70-3.73 (1H, m), 3.80 (1H, dd, J=11.4,
5.1 Hz), 4.08-4.15 (3H, m); .sup.13C-NMR (75 MHz, CDCl.sub.3)
.delta. 18.8, 19.0, 22.2, 22.3, 22.5, 23.7, 24.2, 29.0, 31.8, 34.5,
37.3, 37.6, 38.0, 50.9, 58.8, 70.6, 72.0, 80.5, 101.0; HRMS calc'd
for C.sub.19H.sub.36O.sub.4: 328.2614; found: 328.2619;
[.alpha.].sup.26.0.sub.d:-0.78.degree. (c 5.48,
CH.sub.2Cl.sub.2).
Formula 511
[0311] To crude diol 510 was added pyridine (316 .mu.L, 3.91 mmol)
and chloroacetic anhydride (229 mg, 1.3 mmol) at -78.degree. C. and
stirred for 2 hr. Saturated aqueous sodium bicarbonate (10 mL) was
added and the layers were separated. The aqueous layer was
extracted with methylene chloride (3.times.5 mL). The combined
organics were dried over sodium sulfate and the solvent was removed
in vacuo. Chromatography (10%-20%-30% EtOAc/pentane) provided
residual starting material A61 (89.8 mg, 31%) and chloroacetate
ester 511 (152.5 mg, 44%). 511: R.sub.f (20% EtOAc/pentane)=0.56;
IR (film): 3515.2, 2954.4, 2871.9, 1738.5, 1455.8, 1371.6, 1308.7,
1132.8, 972.7 cm.sup.-1; .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta.
0.75 (1H, t, J=13.2 Hz), 0.87 (3H, d, J=2.7 Hz), 0.89 (3H, d, J=3
Hz), 0.92-0.93 (10H, m), 1.19-1.26 (2H, m), 1.39-1.79 (7H, m), 2.40
(1H, quin, J=6.9 Hz), 2.74 (1H, br. d, J=13.2 Hz), 3.65-3.66 (2H,
m), 3.81 (1H, dd, J=11.6, 4.7 Hz), 4.00-4.17 (4H, m), 4.07 (2H, s);
.sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. 18.9, 19.3, 21.4, 22.2,
22.3, 23.8, 24.2, 29.0, 31.8, 34.5, 37.3, 37.4, 38.2, 40.9, 50.9,
58.8, 70.5, 71.6, 76.1, 101.0, 167.3; HRMS calc'd for
C.sub.11H.sub.37ClO.sub.5: 404.2330; found: 404.2329;
[.alpha.].sup.24.3.sub.d:+4.95.degree. (c 3.43,
CH.sub.2Cl.sub.2).
Formula 512
[0312] To acid 28 (structure not shown) prepared as described in
Theisen et al. (1998) (hydrogen (3R, 1'R)-1-(1'-naphthyl)ethyl
3-[(tert-butyldimethylsilyl)oxy]pentanedioate, 309 mg, 0.744 mmol)
in toluene (6 mL) was added triethylamine (263 .mu.L, 1.98 mmol)
followed by the Yamaguchi reagent (124 .mu.L, 0.79 mmol) and
stirred for 2 hr at room temperature. DMAP (303 mg, 2.50 mmol) was
then added followed by 511 (190 mg, 0.469 mmol) and the mixture was
stirred for 45 min. The reaction was then diluted with EtOAc (5 mL)
and saturated aqueous sodium bicarbonate (10 mL) was added. The
layers were separated and the aqueous layer was extracted with
EtOAc (3.times.5 mL). The combined organics were washed with
saturated aqueous ammonium chloride (10 mL), the layers were
separated and the aqueous layer was extracted with EtOAc (2.times.5
mL). The combined organics were dried over sodium sulfate and the
solvent was removed in vacuo. Chromatography (5%-10% EtOAc/pentane)
provided 512 (311.9 mg, 83%): R.sub.f (10% EtOAc/pentane)=0.46; IR
(film): 2953.8, 2868.2, 1738.1, 1471.9, 1373.6, 1307.9, 1258.5,
1160.3, 1108.7, 1069.3, 977.5, 837.0, 777.9 cm.sup.-1; .sup.1H-NMR
(500 MHz, CDCl.sub.3) .delta. 0.04 (3H, s), 0.12 (3H, s), 0.61 (1H,
t, J=13.2 Hz), 0.79 (9H, s), 0.82-0.92 (9H, m), 0.94 (3H, s), 0.95
(3H, s), 1.12-1.15 (1H, m), 1.35-1.47 (1H, m), 1.56-1.65 (4H, m),
1.69 (3H, d, J=6.5 Hz), 1.72-1.79 (1H, m), 2.37 (1H, quin, J=6.9
Hz), 2.56-2.63 (4H, m), 2.68 (1H, dd, J=15.3, 5.3 Hz), 3.64-3.68
(1H, m), 3.76 (1H, dd, J=11.8, 4.8 Hz), 3.86 (1H, d, J=11 Hz), 3.97
(1H, td, J=12.6, 1.8 Hz), 4.01 (1H, d, J=11 Hz), 4.05 (2H, s), 4.51
(1H, quin, J=6 Hz), 4.98 (1H, dd, J=9.5, 1.0 Hz), 6.64 (1H, q,
J=6.5 Hz), 7.42-7.53 (3H, m), 7.57 (1H, d, J=7.0 Hz), 7.78 (1H, d,
J=8.0 Hz), 7.85 (1H, d, J=8.5 Hz), 8.06 (1H, d, J=8.5 Hz));
.sup.13C-NMR (125 MHz, CDCl.sub.3) .delta. -5.1, -4.8, 14.2, 17.8,
19.0, 20.3, 21.4, 21.7, 21.8, 22.3, 23.7, 24.3, 25.6, 28.8, 31.0,
34.9, 37.2, 37.3, 38.1, 40.7, 41.8, 42.1, 51.1, 58.9, 65.6, 65.7,
69.7, 70.9, 73.0, 100.5, 123.1, 123.2, 125.3, 125.6, 126.2, 128.4,
128.8, 130.1, 133.8, 137.3, 167.1, 170.1, 170.4; HRMS calc'd for
C.sub.44H.sub.67ClO.sub.9Si (+1Na): 825.4149; found: 825.4141;
[.alpha.].sup.25.5.sub.d:-2.17.degree. (c 8.42,
CH.sub.2Cl.sub.2).
Formula 513
[0313] To 512 (156 mg, 0.194 mmol) in EtOAc (4.4 mL) at room
temperature was added Pd(OH).sub.2/C (75 mg). The black slurry was
stirred and the flask was evacuated and refilled with hydrogen (5
times). After 5.5 h under 1 atm. of hydrogen, the reaction was
poured directly onto a silica column pre-packed with pentane and
eluted (20% EtOAc+1% AcOH/pentane) to provide 119 mg of linker
synthon 513 in 95% yield. 513: R.sub.f(20% EtOAc+1%
AcOH/pentane)=0.27; IR (film): 2800.0-3422.4, 2954.1, 2866.6,
1738.3, 1714.1, 1473.1, 1375.8, 1308.0, 1258.4, 1159.6, 1107.3,
977.0, 837.3, 778.8 cm.sup.-1; .sup.1H-NMR (300 MHz, CDCl.sub.3)
.delta. 0.08 (3H, s), 0.09 (3H, s), 0.66 (1H, t, J=13.0 Hz),
0.85-0.92 (19H, m), 0.95 (3H, s), 0.97 (3H, s), 1.06-1.25 (1H, m),
1.33-1.50 (4H, m), 1.58-1.81 (4H, m), 2.37 (1H, dsept, J=6.6, 1.2
Hz), 2.53 (1H, dd, J=15.3, 6.9 Hz), 2.58-2.60 (3H, m), 2.66 (1H,
dd, J=15.0, 4.8 Hz), 3.64-3.68 (1H, m), 3.79 (1H, dd, J=11.7, 4.2
Hz), 3.86 (1H, d, J=11.1 Hz), 3.98-4.03 (1H, m), 4.00 (1H, d,
J=11.1 Hz), 4.06 (2H, s), 4.49 (1H, quin, J=5.9 Hz), 4.98 (1H, dd,
J=9.6, 1.8 Hz)); .sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. -5.1,
-4.8, 17.9, 19.0, 20.3, 21.4, 21.7, 22.3, 23.7, 24.3, 25.7, 28.8,
31.1, 34.9, 37.3, 37.4, 38.2, 40.7, 41.9, 42.0, 51.1, 58.9, 65.7,
65.7, 70.9, 73.3, 100.6, 167.2, 170.2, 176.4; HRMS calc'd for
C.sub.32H.sub.57ClO.sub.9 Si (+1Na): 671.3348; found: 671.3358;
[.alpha.].sup.27.4.sub.d:-20.46.degree. (c 7.46,
CH.sub.2Cl.sub.2).
Example 3
Exemplary Bryostatin Analogues
3A. Formula II--C26 Des-Methyl Bryostatin Analogue (702)
[0314] ##STR59##
201.1 (Formula 201 where R.sup.20 is --O--CO--C.sub.7H.sub.15 and
R.sup.21 is .dbd.CH--CO.sub.2Me)
[0315] To di-benzyl ether 111.1 (Example 1C, 503 mg, 0.169 mmol) in
EtOAc (12.3 mL) at room temperature is added Pd(OH).sub.2/C (82
mg). The black slurry was stirred vigorously and the flask was
evacuated and refilled 4 times with hydrogen (1 atm). After 1 h,
the reaction was poured directly onto a silica column and eluted
(50% EtOAc/pentane to 100% EtOAc) to provide 201.1 (240.2 mg, 65%)
as a colorless oil.
[0316] 201.1: R.sub.f(50% EtOAc/pentane)=0.36; IR (film)=3424,
2955, 2930, 2857, 1748, 1722, 1156, 1081, 837, 776 cm.sup.-1;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.00 (6H, s), 0.84-0.93
(12H, m), 0.96 (3H, s), 0.98 (3H, s), 1.20 (3H, d, J=6.0 Hz),
1.15-1.38 (10H, m), 1.62 (1H, t, J=7.1 Hz), 1.71 (1H, t, J=5.7 Hz),
2.25-2.45 (2H, m), 2.54 (1H, s), 2.89 (1H, d, J=4.2 Hz), 3.34-3.48
(1H, m), 3.36 (3H, s), 3.52 (2H, dd, J=15.9, 9.6 Hz), 3.58-3.76
(3H, m), 3.67 (3H, s), 4.14-4.26 (1H, m), 5.55 (1H, s), 5.89 (1H,
s); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. -5.5, 14.0, 18.4,
19.4, 20.7, 22.5, 24.6, 25.9, 28.8, 29.0, 31.6, 32.4, 34.4, 39.0,
46.6, 51.1, 51.3, 67.5, 68.4, 70.9, 71.9, 72.3, 103.1, 116.9,
152.4, 166.5, 171.8; [.alpha.].sup.23.9.sub.D=-2.24.degree.
(c=6.53, CH.sub.2Cl.sub.2). ##STR60##
202.1 (Formula 202 where R.sup.20 is --O--CO--C.sub.7H.sub.15 and
R.sup.21 is .dbd.CH--CO.sub.2Me)
[0317] To diol 201.1 (26.8 mg, 0.045 mmol) in benzene (1.2 mL)
under nitrogen at 0.degree. C. was added triethylamine (30 .mu.L,
0.227 mmol) followed by lead tetraacetate (50 mg, 0.113 mmol). The
resulting suspension was stirred at 0.degree. C. for 20 min. and
was then quenched with an aqueous solution of saturated ammonium
chloride (5 mL) and extracted with ethyl acetate (3.times.5 mL).
The combined organic layers were dried over sodium sulfate, the
solution was decanted and then the solvent was removed in vacuo to
provide the crude aldehyde (structure not shown) which was taken
immediately to the next step.
[0318] To the crude aldehyde (34 mg, 0.061 mmol) in THF (1.4 mL)
under nitrogen at 0.degree. C. was added a 0.5M solution of the
Tebbe reagent in toluene (122 .mu.L, 0.061 mmol) dropwise. The
reddish-black slurry was stirred at 0.degree. C. for 15 min. and
was then quenched with a saturated aqueous solution of sodium
bicarbonate (5 mL). The biphasic mixture was extracted with ethyl
acetate (3.times.5 mL). The combined organic layers were dried over
sodium sulfate, the solution was decanted and then the solvent was
removed in vacuo. Chromatography (5% EtOAc/pentane) provides olefin
202.1 (19 mg, 56%-2 steps) as a colorless oil.
[0319] 202.1: R.sub.f (10% EtOAc/pentane)=0.62; IR (film)=2955,
2930, 2857, 1747, 1722, 1667, 1155, 1082, 837, 775 cm.sup.-1;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.01 (6H, s), 0.80-0.90
(12H, m), 0.96 (3H, s), 1.00 (3H, s), 1.20-1.38 (10H, m), 1.56-1.72
(1H, m), 2.24-2.46 (4H, m), 3.43 (1H, d, J=15.9 Hz), 3.30 (3H, s),
3.54 (2H, dd, J=18.9, 9.3 Hz), 3.68 (3H, s), 3.87-3.91 (1H, m),
5.09-5.16 (2H, m), 5.53 (1H, s), 5.80-5.98 (1H, m), 5.88 (1H, s);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. -5.4, 14.0, 18.4, 20.5,
20.6, 22.6, 24.7, 25.9, 28.9, 29.0, 31.6, 32.1, 34.5, 40.0, 47.0,
51.1, 67.3, 71.0, 72.1, 103.0, 116.6, 117.7, 133.8, 153.1, 166.6,
171.9; [.alpha.].sup.22.0.sub.D=-7.91.degree. (c=1.91,
CH.sub.2Cl.sub.2). ##STR61##
203.1 (Formula 203 where R.sup.20 is --O--CO--C.sub.7H.sub.15 and
R.sup.21 is .dbd.CH--CO.sub.2Me)
[0320] To a solution of silyl ether 202.1 (101.7 mg, 0.1833 mmol)
and pyridine (267 .mu.L, 3.30 mmol) in THF (1.53 mL) in a
polypropylene vial was added 70% HF/pyridine complex (104.8 .mu.L,
3.67 mmol) at room temperature. The solution was stirred for 18
hours and was then quenched with a saturated aqueous solution of
sodium bicarbonate (5 mL). The biphasic mixture was extracted with
ethyl acetate (4.times.5 mL). The combined organic layers were
dried over sodium sulfate, the solution was decanted, and the
solvent was removed in vacuo to afford the corresponding
de-silylated alcohol (structure not shown) as a pale yellow oil
which was used immediately in the next step.
[0321] The crude alcohol was dissolved in CH.sub.2Cl.sub.2 (2 mL)
and treated with a single portion of
1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (Dess-Martin
periodinane, 117 mg, 0.275 mmol) at room temperature. The mixture
was stirred for 45 min and quenched with saturated aqueous
NaHCO.sub.3/Na.sub.2S.sub.2O.sub.3 (2 mL). The two phase system was
vigorously stirred until the organic layer has cleared (90 min).
The layers were then separated and the aqueous phase was extracted
with CH.sub.2Cl.sub.2 (4.times.5 mL). The combined organic layers
were dried over sodium sulfate, the solution was decanted and then
the solvent was removed in vacuo. Chromatography on silica gel
(7.5% EtOAc/hexanes) provides aldehyde 203.1 (43.7 mg, 54%-2 steps)
as a colorless oil.
[0322] 203.1: R.sub.f(15% EtOAc/pentane)=0.58; IR (film)=2930,
2857, 1750, 1723, 1668, 1160, 1048 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 0.86 (3H, t, J=6.3 Hz, octanoate Me), 1.00 (3H,
s, C18 Me), 1.16 (3H, s, C18 Me), 1.18-1.31 (10H, m), 1.46-1.60
(2H, m), 2.18 (2H, t, J=7.4 Hz), 2.43 (2H, t, J=6.6 Hz), 3.39 (3H,
s), 3.66 (1H, d, J=18.9 Hz), 3.69 (3H, s), 3.74-3.84 (1H, m),
5.13-5.20 (2H, m), 5.85-6.00 (1H, m), 5.96 (1H, s), 9.71 (1H, s);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.1, 16.3, 19.1, 22.6,
24.3, 28.9, 30.4, 31.6, 38.9, 40.0, 51.2, 51.4, 54.0, 71.5, 71.9,
102.2, 118.2, 119.8, 133.4, 150.3, 166.4, 171.7, 202.4;
[.alpha.].sup.23.6.sub.D=-6.07.degree. (c=2.23, CH.sub.2Cl.sub.2).
##STR62##
204.1 (Formula 204 where R.sup.20 is --O--CO--C.sub.7H.sub.15 and
R.sup.21 is .dbd.CH--CO.sub.2Me)
[0323] A dihydroxylating stock solution was generated by dissolving
(DHQD).sub.2AQN (3.6 mg, 0.00425 mmol), K.sub.3Fe(CN).sub.6 (425
mg, 1.275 mmol), K.sub.2CO.sub.3 (175 mg, 1.275 mmol) and
K.sub.2OsO.sub.2 (OH).sub.4 (0.65 mg, 0.00175 mmol) in t-BuOH (2.1
mL) and water (2.1 mL). The resulting solution was stirred at room
temperature for 3 h. 504 .mu.L of this stock solution was added to
olefin 203.1 (7.4 mg, 0.017 mmol) pre-dissolved in t-BuOH (200
.mu.L) and water (200 .mu.L) under nitrogen at 0.degree. C. The
resulting solution was stirred at 0-5.degree. C. for 2 days. Water
(2 mL) was then added and the biphasic mixture was then extracted
with EtOAc (4.times.5 mL). The combined organic layers were dried
over sodium sulfate, the solution was decanted and then the solvent
was removed in vacuo. Chromatography (90% EtOAc/pentane to 100%
EtOAc) provides diol 204.1 (5.6 mg, 70%) as an approximately 2.2:1
(.beta.:.alpha.) mixture of diastereomers as a colorless oil.
[0324] 204.1: R.sub.f(90% EtOAc/pentane)=0.36; IR (film)=3418,
2932, 2860, 1750, 1722, 1668, 1159, 1081, 1052 cm.sup.-1; .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 0.86 (3H, t, J=6.8 Hz), 1.01 (3H,
s, major), 1.02 (3H, s, minor), 1.15 (3H, s, minor), 1.17 (3H, s,
major), 1.20-1.36 (10H, m), 1.47-1.61 (1H, m), 1.70-1.85 (1H, m),
2.10-2.32 (3H, m), 2.55 (1H, br. s, major), 3.09 (1H, br. s,
minor), 3.42-3.80 (4H, m), 3.45 (3H, s, minor), 3.46 (3H, s,
major), 3.69 (3H, s), 3.98-4.20 (2H, m), 5.20 (1H, s, major), 5.25
(1H, s, minor), 5.96 (1H, s), 9.68 (1H, s, minor) 9.68 (1H, s,
major); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 14.0, 16.5,
16.7, 19.0, 19.1, 22.5, 24.3, 28.9, 31.3, 31.3, 31.6, 33.9, 38.8,
38.9, 51.3, 51.3, 51.5, 51.6, 53.9, 66.7, 67.2, 68.2, 68.7, 70.3,
71.0, 71.4, 71.7, 102.2, 102.6, 119.6, 119.7, 150.0, 166.3, 171.7,
183.9, 201.8, 202.2. ##STR63##
205.1 (Formula 205 where R.sup.20 is --O--CO--C.sub.7H.sub.15 and
R.sup.21 is .dbd.CH--CO.sub.2Me)
[0325] To diol 204.1 (28.5 mg, 0.0603 mmol) in methylene chloride
(2.45 mL) was added pyridine (141.3 .mu.L, 1.75 mmol) followed by
TESCl (176 mL, 1.05 mmol) at room temperature. The resulting clear
solution was stirred at room temperature for 15 h. Triethylamine
(250 .mu.L) was then added and the solution was directly loaded
onto a silica column and eluted (5% EtOAc+5% triethylamine/pentane)
to afford bis silyl ether 205.1 (42.2 mg, 100%) as an approximately
2.2:1 (.beta.:.alpha.) mixture of diastereomers as a colorless
oil.
[0326] 205.1: R.sub.f (5% EtOAc+5% triethylamine/pentane)=0.58; IR
(film)=2955, 2877, 1754, 1724, 1668, 1462, 1231, 1159, 1107, 1007,
743 cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.44-0.65
(12H, m), 0.86 (3H, t, J=6.8 Hz), 0.88-0.98 (18H, m), 0.98 (3H, s,
major), 1.00 (3H, s, minor), 1.14 (3H, s, major), 1.15 (3H, s,
minor), 1.18-1.32 (110H, m), 1.58-1.71 (1H, m), 1.82-2.00 (1H, m),
2.09-2.21 (3H, m), 3.30-3.71 (3H, m), 3.41 (3H, s, minor), 3.43
(3H, s, major), 3.68 (3H, s, minor), 3.69 (3H, s, major), 3.90-4.10
(2H, m), 5.19 (1H, s, minor), 5.22 (1H, s, major), 5.94 (1H, s,
minor), 6.00 (1H, s, major), 9.69 (1H, s, major) 9.70 (1H, s,
minor); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 4.3, 4.3, 4.9,
5.2, 6.2, 6.7, 6.8, 6.9, 14.0, 16.4, 16.5, 18.9, 19.0, 22.5, 24.3,
28.9, 29.7, 31.4, 31.6, 31.8, 33.9, 41.2, 41.5, 51.1, 51.2, 51.4,
51.7, 53.9, 67.3, 67.5, 68.0, 69.3, 69.8, 70.3, 71.1, 71.3, 102.1,
102.3, 119.4, 119.5, 150.2, 150.3, 151.0, 166.2, 166.2, 171.7,
202.5, 202.6. ##STR64##
206.1 (Formula 206 where R.sup.20 is --O--CO--C.sub.7H.sub.15 and
R.sup.21 is .dbd.CH--CO.sub.2Me)
[0327] To a solution of diethylmethoxyborane (361 .mu.L, 2.75 mmol)
in Et.sub.2O (2.14 mL) was added allylmagnesium bromide (1.0M in
Et.sub.2O, 2.50 mmol, 2.50 mL) dropwise at 0.degree. C. The white
precipitous mixture was stirred at 0.degree. C. for 60 min. and
then allowed to stand for 5 min. A 41.6 .mu.L aliquot (0.5 M
allyldiethylborane, 0.021 mmol) of this solution was added dropwise
to aldehyde 205.1 (7.3 mg, 0.01 mmol) in 0.5 mL Et.sub.2O at
-10.degree. C. After stirring for 30 min., the reaction was
quenched with aqueous saturated NH.sub.4Cl (5 mL). The biphasic
mixture was then extracted with EtOAc (3.times.5 mL). The combined
organic layers were dried over sodium sulfate, the solution was
decanted and then the solvent was removed in vacuo to provide a
colorless oil which was taken directly onto the next step.
[0328] The crude residue was dissolved in CH.sub.2Cl.sub.2 (1 mL)
and treated successively with triethylamine (17.4 .mu.L, 0.125
mmol), 4-dimethylaminopyridine (15.3 mg, 0.125 mmol) and Ac.sub.2O
(6 .mu.L, 0.06 mmol) at room temperature. The solution was stirred
for 17 h and then pipetted directly onto a short column of silica
gel and the products eluted with 7.5% EtOAc/hexanes to afford a
diastereomeric mixture of homoallylic acetates (7.9 mg, 97%-2
steps) as a colorless oil.
[0329] A portion of the isolated homoallylic acetate (5 mg, 0.0064
mmol) was dissolved in THF (253 .mu.L) and H.sub.2O (25.3 .mu.L)
and treated with N-methylmorpholine N-oxide (1.6 mg, 0.014 mmol)
followed by OsO.sub.4 (4 wt % in H.sub.2O-16 .mu.L, 0.0025 mmol) at
room temperature. The homogeneous solution was stirred for 3 h, and
then aqueous saturated sodium bicarbonate (4 mL) and water (1 mL)
was added. The biphasic mixture was extracted with EtOAc (5.times.5
mL). The combined organic layers were dried over sodium sulfate,
the solution was decanted and then the solvent was removed in vacuo
to provide a colorless oil which was taken directly to the next
step.
[0330] The resulting residue was immediately dissolved in benzene
(0.4 mL) and treated with Et.sub.3N (2.6 .mu.L, 0.025 mmol) at room
temperature. Solid Pb(OAc).sub.4 (4.2 mg, 0.0096 mmol) was quickly
added in one portion and the resulting yellow precipitous mixture
was stirred vigorously for 30 min.
1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 12 .mu.L, 0.08 mmol) was
introduced and the reaction mixture was stirred for another 30 min.
The mixture was added directly to a silica column and eluted (10%
EtOAc/Pet. ether) to provide unsaturated aldehyde 206.1 (3.4 mg,
73%-2 steps) as an approximately 2.2:1 (.beta.:.alpha.) mixture of
diastereomers as a colorless oil.
[0331] 206.1: R.sub.f(10% EtOAc/Pet. ether)=0.42; IR (film)=2955,
2877, 1748, 1723, 1692, 1461, 1229, 1154, 1105, 1008, 743
cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.55-0.68
(12H, m), 0.87 (3H, t, J=6.8 Hz), 0.92-1.02 (18H, m), 1.15 (3H, s),
1.18 (3H, s), 1.20-1.34 (10H, m), 1.48-1.60 (2H, m), 1.85-2.03 (1H,
m), 2.04-2.23 (2H, m), 2.27-2.39 (1H, m), 3.45-3.68 (2H, m), 3.38
(3H, s, minor), 3.40 (3H, s, major), 3.69 (3H, s, minor), 3.70 (3H,
s, major), 3.95-4.05 (1H, m), 4.06-4.17 (1H, m), 5.45 (1H, s,
minor), 5.47 (1H, s, major), 5.89 (1H, s), 5.93 (1H, dd, J=16.1/7.8
Hz, major), 5.93 (1H, dd, J=16.1/7.8 Hz, minor), 7.33 (1H, d,
J=16.1 Hz, major), 7.36 (1H, d, J=16.1 Hz, minor), 9.53 (1H, d,
J=7.8 Hz, major), 9.54 (1H, d, J=7.8 Hz, minor); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 4.3, 4.4, 4.9, 5.4, 6.7, 6.8, 6.9, 7.0,
14.0, 21.7, 21.8, 22.5, 23.6, 23.8, 24.5, 28.9, 28.9, 31.6, 32.5,
32.8, 34.4, 41.2, 41.6, 47.3, 51.2, 51.4, 51.5, 67.3, 67.6, 68.9,
69.2, 69.8, 70.3, 71.0, 71.1, 102.4, 102.6, 117.6, 117.7, 126.7,
151.6, 166.2, 167.2, 171.7, 194.6. ##STR65##
207.1b (Formula 207 where R.sup.20 is --O--CO--C.sub.7H.sub.15 and
R.sup.21 is .dbd.CH--CO.sub.2Me)
[0332] Enal 206.1 (22 mg, 0.03 mmol) was dissolved in acetonitrile
(2 mL) and water (205 .mu.L) at room temperature. 48% aqueous HF
(388 .mu.L, 12.1 mmol) was added dropwise and the resulting clear
solution was stirred at room temperature for 75 min. and was then
quenched with a saturated aqueous solution of sodium bicarbonate
(15 mL) and water (3 mL). The mixture was extracted with ethyl
acetate (5.times.10 mL). The combined organic layers were dried
over sodium sulfate, the solution was decanted and then the solvent
was removed in vacuo to provide a crude diol which was taken
immediately to the next step.
[0333] A 0.75 mM silylating solution was generated by the addition
of imidazole (62 mg, 0.91 mmol) and TBSCl (45.6 mg, 0.3 mmol) to
methylene chloride (3.9 mL) at room temperature under nitrogen. To
the crude diol dissolved in methylene chloride (3.9 mL) and DMF
(0.4 mL) was added the above stock solution (1 mL) and stirred at
room temperature for 2 h. The solution was then quenched with an
aqueous solution of saturated ammonium chloride (10 mL) and
extracted with methylene chloride (4.times.10 mL). The combined
organic layers were further washed with brine (10 mL) and then
dried over sodium sulfate. The solution was decanted and then the
solvent was removed in vacuo. Chromatography (40% EtOAc/pentane)
provides the silylated C25 .beta. isomer 207.1b (10.4 mg, 57.4%)
along with the silylated C25 .alpha. isomer 207.1a (4.6 mg, 25.4%)
as colorless oils.
[0334] 207.1b: R.sub.f(40% EtOAc/pentane)=0.38; IR (film)=3421,
2930, 2857, 1723, 1691, 1257, 1156, 1110, 837, 779 cm.sup.-1;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.10 (6H, s), 0.87 (3H,
t, J=6.8 Hz), 0.92 (9H, s), 1.14 (3H, s), 1.15 (3H, s), 1.18-1.32
(10H, m), 1.49 (1H, t, J=7.2 Hz), 1.66-1.74 (1H, m), 1.84-2.00 (1H,
m), 1.98-2.15 (3H, m), 3.49 (1H, dd, J=10.0, 6.0 Hz), 3.66-3.76
(1H, m), 3.70 (3H, s), 3.82-4.00 (2H, m), 4.07 (1H, t, J=6.6 Hz),
4.20 (1H, t, J=10.8 Hz), 5.13 (1H, s), 5.96 (1H, dd, J=16.0/7.7
Hz), 6.03 (1H, s), 7.35 (1H, d, J=16.0 Hz), 9.57 (1H, d, J=7.7 Hz);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. -5.3, 14.0, 18.4, 20.0,
22.5, 23.1, 24.4, 25.9, 28.9, 28.9, 31.1, 31.6, 34.5, 39.0, 45.7,
51.3, 67.1, 67.2, 67.9, 72.6, 99.7, 120.7, 127.6, 150.1, 166.1,
171.7, 194.5; [.alpha.].sup.26.6.sub.D=-27.24.degree. (c=1.04,
CH.sub.2Cl.sub.2). ##STR66##
701.1 (Formula 701 where R.sup.20 is --O--CO--C.sub.7H.sub.15,
R.sup.21 is .dbd.CH--CO.sub.2Me and R.sup.26 is H)
[0335] To a solution of acid 408 (Example 2B, 11.2 mg, 0.02 mmol)
in toluene (0.9 mL) was added 2,4,6-trichlorobenzoyl chloride (3.2
.mu.l, 0.02 mmol) at room temperature and the mixture was stirred
for 45 min. Alcohol 207.1b (10.2 mg, 0.017 mmol) and DMAP (10.4 mg,
0.085 mmol) in toluene (1.4 mL) were added and stiffed for 30 min.
The mixture was directly loaded onto a silica gel column and eluted
(7.5% EtOAc/pentane) to afford the ester 701.1 (15.2 mg, 79%).
[0336] 701.1: R.sub.f (15% ethyl acetate/pentane)=0.29; IR
(film)=3492, 2930, 2859, 1725, 1691, 1155, 1112, 837, 777
cm.sup.-1; 111 NMR (400 MHz, CDCl.sub.3) .delta. 0.04 (3H, s), 0.05
(3H, s), 0.07 (6H, s), 0.68 (1H, t, J=13.2 Hz), 0.60-0.92 (30H, m),
1.10-1.30 (18H, m), 1.34-1.84 (18H, m), 1.85-2.14 (3H, m),
2.34-2.52 (3H, m), 2.68 (1H, d, J=12.3 Hz), 3.13 (1H, s), 3.34-3.46
(2H, m), 3.60-3.70 (3H, m), 3.68 (3H, s), 3.76-3.86 (2H, m),
3.86-3.96 (1H, m), 4.06 (1H, dt, J=11.9, 2.1 Hz), 4.24-4.34 (1H,
m), 5.11 (1H, s), 5.20-5.32 (1H, m), 5.96 (1H, dd, J=16.1, 7.8 Hz),
6.00 (1H, s), 7.42 (1H, d, J=16.1 Hz), 9.58 (1H, d, J=7.8 Hz);
.sup.13C NMR (1100 MHz, CDCl.sub.3) .delta. -5.3, -5.3, -4.7, -4.6,
14.0, 18.0, 18.3, 19.2, 20.1, 21.9, 22.3, 22.5, 22.9, 23.6, 23.7,
23.8, 24.3, 24.5, 25.8, 28.9, 28.9, 29.2, 30.9, 31.0, 31.6, 31.8,
31.9, 32.0, 34.5, 35.0, 37.4, 37.4, 43.5, 43.8, 44.7, 45.6, 51.2,
51.3, 59.2, 64.4, 64.9, 65.7, 66.3, 71.1, 72.7, 73.4, 73.8, 99.5,
100.4, 120.5, 127.5, 150.5, 166.4, 166.5, 171.7, 172.1, 194.6;
[.alpha.].sup.24.4.sub.D=-27.42.degree. (c=0.87, CH.sub.2Cl.sub.2).
##STR67##
702.1 (Formula 702 where R.sup.3 is OH, R.sup.20 is
--O--CO--C.sub.7H.sub.15, R.sup.21 is .dbd.CH--CO.sub.2Me and
R.sup.26 is H)
[0337] To seco aldehyde 701.1 (15 mg, 0.013 mmol) in THF (3.7 mL)
at room temperature in a plastic flask was added 70% HF/pyridine
dropwise. The resulting yellow solution was stirred for 2 hr and
was then quenched with a saturated aqueous solution of sodium
bicarbonate (12.5 mL) and water (7.5 mL). The biphasic mixture was
extracted with ethyl acetate (5.times.15 mL). The combined organic
layers were dried over sodium sulfate, the solution was decanted
and then the solvent was removed in vacuo. Chromatography (70%
EtOAc/pentane) provides 702.1 (7 mg, 73%) (the corresponding
compound of Formula II where X is oxygen) as an amorphous
solid.
[0338] 702.1: R.sub.f (80% EtOAc/pentane)=0.29; IR (film)=3454,
3332, 2932, 2858, 1723, 1663, 1138, 976 cm.sup.-1; .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 0.86 (3H, t, J=6.7 Hz), 1.01 (3H, s), 1.17
(3H, s), 1.18-1.38 (12H, m), 1.40-1.66 (7H, m), 1.72-1.88 (3H, m),
1.94-2.14 (3H, m), 2.29 (1H, dt, J=7.5/1.7 Hz), 2.52 (1H, d, J=7.2
Hz), 3.45 (1H, t, J=11.2 Hz), 3.53 (1H, t, J=10.6 Hz), 3.61-3.72
(4H, m), 3.68 (3H, s), 3.88 (3H, t, J=12.4 Hz), 4.02-4.09 (2H, m),
4.10-4.19 (1H, m), 4.48 (1H, d, J=11.6 Hz), 5.02 (1H, d, J=7.6 Hz),
5.10 (1H, s), 5.13 (1H, s), 5.34-5.39 (1H, m), 5.40 (1H, dd,
J=15.0/7.6 Hz), 5.97 (1H, d, J=15 Hz), 5.99 (1H, s); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. 14.1, 19.3, 22.5, 23.0, 24.4, 24.7,
28.9, 29.0, 31.0, 31.3, 31.4, 31.6, 32.4, 34.6, 35.9, 39.9, 42.5,
42.9, 45.1, 51.1, 64.5, 65.8, 66.3, 68.6, 71.6, 74.1, 75.8, 76.0,
78.7, 98.9, 102.4, 119.9, 125.7, 142.6, 151.7, 167.0, 172.1, 172.6;
[.alpha.].sup.24.0.sub.D=-20.02.degree. (c=0.70,
CH.sub.2Cl.sub.2).
3B. Formula IV--Bryostatin Analogue Containing Ether Diester Linker
(807)
[0339] ##STR68##
801.1 and 802.1 (Formulae 801 and 802 where R.sup.20 is
--O--CO--C.sub.7H.sub.15 and R.sup.21 is .dbd.CH--CO.sub.2Me)
[0340] Enal 801.1, prepared as described for compound 13 in Wender
et al. (1998a) (22 mg, 0.030 mmol) in a tert-butanol-THF solution
of 2-methyl 2-butene (1:1, 3 mL) was treated with sodium chlorite
(14 mg, 0.152 mmol) and monobasic sodium phosphate (21 mg, 0.152
mmol) in water (0.5 mL). After 1 h, the mixture was diluted with
ethyl acetate. The organic layer was dried over sodium sulfate.
Column chromatography afforded acid 802.1: R.sub.f=(25% ethyl
acetate in hexane); [.alpha.].sup.25.sub.D=-5.97.degree. (c 0.40,
CH.sub.2Cl.sub.2); IR (neat)=2932, 1716, 1644, 1514, 1456, 1374
cm.sup.-1; .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.59 (d,
J=16.0 Hz, 1H), 7.38 (m, 5H), 7.18 (d, J=8.5 Hz, 2H), 6.85 (d,
J=8.5 Hz, 2H), 5.88 (s, 1H), 5.24 (d, J=16.0 Hz, 1H), 5.51 (s, 1H),
5.66 (d, J=11.0 Hz, 1H), 5.60 (d, J=11.0 Hz, 2H), 5.39 (d, J=11.0
Hz, 1H), 4.13 (br, 1H), 3.97 (m, 1H), 3.85 (m, 1H), 3.81 (s, 3H),
3.70 (s, 3H), 3.49 (d, J=14.5 Hz, 1H), 3.26 (s, 3H), 2.41 (t,
J=14.5 Hz, 1H), 2.26 (m, 2H), 2.00 (m, 1H), 1.76 (m, 1H), 1.29-1.16
(m, 21H), 0.88 (t, J=7.0 Hz, 3H); .sup.13C NMR (125 MHz,
CDCl.sub.3) .delta. 172.28, 172.05, 166.29, 159.69, 159.10, 152.00,
138.62, 130.51, 129.17, 128.35, 127.61, 127.52, 117.07, 114.72,
113.75, 102.40, 76.19, 74.30, 71.68, 71.11, 70.80, 68.65, 55.23,
51.16, 48.83, 46.87, 36.08, 35.35, 34.13, 32.97, 31.62, 28.89,
24.53, 23.32, 22.50, 22.25, 14.25, 14.07, ##STR69##
803.1 (Formula 803 where R.sup.20 is --O--CO--C.sub.7H.sub.15 and
R.sup.21 is .dbd.CH--CO.sub.2Me)
[0341] Acid 802.1 (22 mg, 0.030 mmol) in toluene (3 mL) was treated
with Yamaguchi's reagent (6:L, 0.0395 mmol) and TEA (16:L, 0.122
mmol). After 30 min, alcohol 606 from Example 2F (17 mg, 0.0395
mmol) and DMAP (11 mg, 0.0912 mmol) in toluene (1 mL) was added and
stirred for 1 h. The mixture was directly purified by silica gel
column to give ester 803.1 (27 mg, 77% yield): R.sub.f=(25% ethyl
acetate in hexane); [.alpha.].sup.25.sub.D=-21.4.degree. (c 0.82,
CH.sub.2Cl.sub.2); IR (neat)=2930, 2858, 1720, 1652, 1612, 1514,
1464, 1383, 1250, 1110 cm.sup.-1; .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 7.46 (d, J=16.0 Hz, 1H), 7.36 (m, 5H), 7.18 (d, J=8.5 Hz,
2H), 6.85 (d, J=8.5 Hz, 2H), 5.87 (s, 1H), 5.67 (d, J=16.0 Hz, 1H),
5.50 (s, 1H), 5.29 (q, J=6.0 Hz, 2H), 4.63 (m, 3H), 4.39 (d, J=11.0
Hz, 1H), 4.29 (t, J=11.0 Hz, 1H), 4.20 (m, 2H), 4.12 (t, J=11.0 Hz,
1H), 3.96 (m, 1H), 3.86.about.3.66 (m, 7H), 3.47 (m, 5H), 3.25 (s,
3H), 2.53 (d, J=6.5 Hz, 2H), 2.39 (br, 1H), 2.24 (m, 2H),
1.99.about.0.85 (m, 26H), 0.09 (s, 3H), 0.07 (s, 3H), 0.04 (s, 9H);
.sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 172.04, 171.07, 167.24,
166.30, 159.10, 157.02, 152.11, 138.68, 130.56, 129.18, 128.35,
128.17, 127.61, 117.04, 115.34, 113.74, 102.42, 88.96, 76.35,
74.48, 71.74, 71.13, 70.90, 68.59, 67.86, 67.34, 67.02, 66.59,
66.61, 61.52, 55.22, 51.12, 46.70, 42.93, 37.24, 36.17, 34.12,
31.64, 29.18, 28.99, 28.88, 25.75, 24.53, 23.45, 22.54, 22.38,
18.00, 17.95, 14.36, 14.06, -1.46, -4.80. ##STR70##
805.1 (Formula 805 where R.sup.20 is --O--CO--C.sub.7H.sub.15 and
R.sup.21 is .dbd.CH--CO.sub.2Me)
[0342] Ester 803.1 (27 mg, 0.0233 mmol) in wet methylene chloride
(2 mL) was treated with DDQ (11 mg, 0.0466 mmol) and stirred for 1
h. The mixture was directly purified by silica gel to give the
expected alcohol silyl ether product of Formula 804. This silyl
ether in acetonitrile-water (10:1, 1.5 mL) was treated with aqueous
HF (100:L, 48%). After 4 h, the mixture was neutralized with
aqueous sodium bicarbonate. The aqueous layer was extracted with
ethyl acetate. The combined organic layer was dried over sodium
sulfate. The crude hydroxy acid product 805.1 was used for the next
step. ##STR71##
807.1 (Formula 807 where R.sup.20 is --O--CO--C.sub.7H.sub.15 and
R.sup.21 is .dbd.CH--CO.sub.2Me)
[0343] To a solution of DCC (13 mg, 0.0623 mmol), DMAP-HCl (10 mg,
0.0623 mmol) and DMAP (11 mg, 0.089 mmol) was added hydroxy acid
805.1 (7 mg, 0.0089 mmol) in methylene chloride (3 mL) by syringe
pump over 10 h. The resultant mixture was loaded directly onto a
silica gel column and purified to give C26-O-benzyl ether lactone
of Formula 806 where R.sup.20 is --O--CO--C.sub.7H.sub.15 and
R.sup.21 is .dbd.CH--CO.sub.2Me (806.1) (3.5 mg, 50%). 806.1:
R.sub.f=(25% ethyl acetate in hexane);
[.alpha.].sup.25.sub.D=4.74.degree. (c 0.47, CH.sub.2Cl.sub.2); IR
(neat)=3746, 3323, 2926, 2851, 1739, 1718, 1624, 1436, 1159
cm.sup.-1; .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.39 (br s,
5H), 6.84 (d, J=16.0 Hz, 1H), 6.03 (s, 1H), 5.80 (d, J=16.0 Hz,
1H), 5.41 (br d, J=12.5 Hz, 1H), 5.20 (s, 1H), 5.15 (s, 1H), 4.64
(q, J=12.0 Hz, 2H), 4.47 (q, J=5.5, 11.5 Hz, 1H), 4.31 (m, 3H),
3.99 (t, J=11.0 Hz, 1H), 3.80-3.43 (m, 8H), 2.52 (m, 2H), 2.37-1.07
(m, 22H), 0.89 (t, J=6.5 Hz, 3H).
[0344] To a solution of benzyl ether 806.1 (1 mg) in methylene
dichloride (0.5 mL) was added boron trichloride (excess) at
-78.degree. C., and the mixture was warmed to -20.degree. C. over 1
h. The reaction mixture was quenched with aqueous sodium
bicarbonate. The standard isolation procedure afforded bryostatin
analogue 807.1. R.sub.f=(25% ethyl acetate in hexane);
[.alpha.].sup.25.sub.D=4.17.degree. (c=0.30, CH.sub.2Cl.sub.2); IR
(neat)=3480, 2927, 2856, 2361, 1718, 1281, 1161, 1105 cm.sup.-1;
.sup.1H NMR (500 MHz, PhH) .delta. 6.82 (d, J=16.5 Hz, 1H), 6.03
(s, 1H), 5.80 (d, J=16.5 Hz, 1H), 5.24 (s, 1H), 5.17 (br s, 2H),
4.36 (m, 3H), 4.01 (t, J=2.5 Hz, 1H), 3.84 (q, J=6.5 Hz, 1H), 3.68
(m, 7H), 3.46 (t, J=9.0 Hz, 1H), 2.57 (m, 2H), 2.33 (m, 2H), 2.18
(m, 1H), 2.11-1.23 (m, 23H), 0.90 (t, J=10.0 Hz, 3H); .sup.13C NMR
(125 MHz, PhH) .delta. 174.37, 172.11, 171.38, 152.76, 151.21,
121.46, 120.31, 119.94, 99.21, 86.71, 73.84, 73.67, 71.96, 70.03,
68.76, 68.38, 65.14, 51.13, 45.48, 41.06, 35.99, 34.58, 32.91,
31.63, 31.14, 29.00, 28.86, 28.65, 24.68, 23.18, 22.55, 21.13,
19.64, 14.05. 3C. Formula III--Bryostatin Analogue Containing
Selected C7 Substituent (705) ##STR72##
704.1 (Formula 704 where R is OH, R' is OBn, R.sup.3 is TBSO,
R.sup.5 is .dbd.O, R.sup.7 is t-Bu-O.sub.2CMeCl, R.sup.8 is H,
R.sup.20 is --O--CO--C.sub.7H.sub.15, R.sup.21 is
.dbd.CH--CO.sub.2Me and R.sup.26 is Me)
[0345] To a methyl hemiacetal prepared as described for compound 14
in Wender et al. (1998a) (30 mg, 0.05 mmol) in acetonitrile (2.8
mL) and water (0.3 mL) was added 48% aq. HF (480 .mu.L) and stirred
at room temp. for 2 hrs. The reaction was then quenched with a
saturated solution of sodium bicarbonate (5 mL) and extracted with
EtOAc (5 mL.times.4). The combined organics were dried over sodium
sulfate and the solvent was removed in vacuo to give expected
hemiacetal (a compound according to Formula 303 where R.sup.20 is
--O--CO--C.sub.7H.sub.15 and R.sup.21 is .dbd.CH--CO.sub.2Me).
[0346] To crude acid 513 from Example 2G (47 mg, 0.075 mmol) in
toluene (2.6 mL) was added triethylamine (27 .mu.L, 0.2 mmol) and
trichlorobenzoyl chloride (12.5 .mu.L, 0.08 mmol), and the mixture
was stirred for 90 min. To the resulting solution is added a
solution containing the crude hemiacetal and DMAP (30.4 mg, 0.25
mmol) in toluene (4 mL). The resulting precipitous mixture was
stirred for 2 h and then added directly to a silica column and
eluted (15% EtOAc/pentane to 25% EtOAc/pentane) to afford seco
aldehyde 704.1 (41.7 mg, 70%): R.sub.f(15% EtOAc/pentane)=0.14; IR
(film): 3497.9, 2953.6, 2867.5, 1738.2, 1688.9, 1469.9, 1378.0,
1258.1, 1158.4, 1109.2, 982.5, 835.5, 779.2, 734.9 cm.sup.-1;
.sup.1H-NMR (500 MHz, CDCl.sub.3) .delta. 0.05 (3H, s), 0.08 (3H,
s), 0.66 (1H, t, J=13.0 Hz), 0.79-0.88 (21H, m), 0.91-0.96 (1H, m),
0.95 (3H, s), 0.97 (3H, s), 1.15 (3H, s), 1.17-1.29 (15H, m),
1.36-1.50 (6H, m), 1.59-1.78 (5H, m), 1.87-2.13 (5H, m), 2.38 (1H,
dquin, J=6.8, 1.3 Hz), 2.51-2.64 (5H, m), 3.31 (1H, s), 3.60-3.68
(2H, m), 3.68 (3H, s), 3.76-3.80 (1H, m), 3.78 (1H, d, J=11.0 Hz),
3.83-3.93 (1H, m), 3.99-4.07 (1H, m), 4.00 (1H, d, J=11.0 Hz), 4.07
(2H, d, J=3.0 Hz), 4.43-4.48 (1H, m), 4.56 (1H, d, J=12.0 Hz), 4.63
(1H, d, J=12.0 Hz), 4.96 (1H, dd, J=9.5, 2.0 Hz), 5.12 (1H, s),
5.41-5.44 (1H, m), 5.95 (1H, dd, J=16.0, 8.0 Hz), 6.00 (1H, d,
J=2.0 Hz), 7.27-7.35 (5H, m), 7.38 (1H, d, J=16.0 Hz), 9.49 (1H, d,
J=8.0 Hz); .sup.13C-NMR (125 MHz, CDCl.sub.3) .delta. -5.1, -4.8,
14.0, 15.0, 17.9, 19.0, 19.8, 20.2, 21.6, 21.7, 22.4, 22.5, 22.8,
23.7, 24.3, 24.4, 25.7, 28.8, 28.8, 28.9, 30.9, 31.1, 31.6, 34.5,
34.9, 35.4, 37.2, 37.5, 38.1, 40.7, 41.6, 42.0, 45.7, 51.1, 51.2,
58.9, 65.6, 65.7, 66.1, 70.8, 71.1, 71.4, 72.5, 73.1, 74.8, 99.5,
100.5, 120.7, 127.5, 127.6, 127.8, 128.4, 138.2, 150.3, 166.2,
166.3, 167.3, 170.5, 171.7, 171.8, 194.6; HRMS calc'd for
C.sub.65H.sub.103ClO.sub.17Si (+1Na): 1241.6532; found: 1241.6551;
[.alpha.].sup.27.0.sub.d:-31.67.degree. (c 4.17, CH.sub.2Cl.sub.2).
##STR73##
705.1 (Formula 705 where R is OH, R' is OBn, R.sup.3 is OH, R.sup.5
is .dbd.O, R.sup.8 is t-Bu-O.sub.2MeCl, R.sup.9 is H, R.sup.20 is
--O--CO--C.sub.7H.sub.15, R.sup.21 is .dbd.CH--CO.sub.2Me and
R.sup.26 is Me)
[0347] To 704.1 (38 mg, 0.031 mmol) in THF (9.5 mL) was added
freshly dried 4 Angstrom molecular sieve beads (57 beads) and 70%
HF/pyridine (2.3 mL), and the resulting solution was stirred for 45
min. in a plastic flask. The reaction was then poured into a
saturated solution of sodium bicarbonate (95 mL) and diluted with
EtOAc (30 mL). The layers were separated and the aqueous layer was
extracted with EtOAc (40 mL.times.3). The combined organic layers
were dried over sodium sulfate and the solvent was removed in
vacuo. Silica chromatography (40% to 100% EtOAc/pentane) afforded
705.1 (13.4 mg, 45%) and a putative diol 705.1a without the
3-hydroxy TBS protecting group (9.0 mg, 30%).
[0348] To putative diol 705.1a (4.8 mg, 4.95 .mu.mol) in THF (0.5
mL) was added freshly dried 4 A molecular sieve beads (3 beads) and
70% HF/pyridine (0.1 mL) and the resulting solution was stirred for
40 min. in a plastic flask. The reaction was then poured into a
saturated solution of sodium bicarbonate (5 mL) and diluted with
EtOAc (30 mL). The layers were separated and the aqueous layer was
extracted with EtOAc (4 mL.times.4). The combined organics were
dried over sodium sulfate and the solvent was removed in vacuo.
Chromatography (30% to 100% EtOAc/pentane) afforded 705.1 (a
compound of Formula III where R.sup.3 is OH, R.sup.5 is .dbd.O,
R.sup.8 is t-Bu-chloroacetate, R.sup.9 is H, R.sup.20 is
--O--CO--C.sub.7H.sub.15, R.sup.21 is .dbd.CH--CO.sub.2Me and
R.sup.26 is Me). (1.1 mg, effectively adding 7% to above
yield=52%). 705.1: R.sub.f (30% EtOAc/pentane)=0.23; IR (film):
3391.9, 2929.4, 2856.8, 1731.9, 1664.5, 1434.2, 1375.1, 1249.6,
1160.5, 1133.2, 1098.3, 982.1, 735.8 cm.sup.-1; .sup.1H-NMR (500
MHz, CDCl.sub.3) .delta. 0.87 (3H, t, J=7.0 Hz), 0.93 (3H, s), 0.96
(3H, s), 1.03 (3H, s), 1.18 (3H, d, J=6.0 Hz), 1.20 (3H, s),
1.22-1.30 (10H, m), 1.51 (1H, br. d, J=12.5 Hz), 1.69-1.81 (3H, m),
2.00-2.08 (4H, m), 2.29-2.34 (3H, m), 2.41 (1H, dd, J=12.5, 4.0
Hz), 2.63 (1H, dd, J=14.3, 2.5 Hz), 2.82 (1H, dd, J=12.5, 4.5 Hz),
3.58 (1H, d, J=11.0 Hz), 3.61-3.64 (1H, m), 3.67-3.74 (2H, m), 3.68
(3H, s), 3.80 (1H, d, J=11.01 Hz), 3.82 (1H, t, J=12.0 Hz),
3.95-3.99 (1H, m), 3.97 (1H, d, J=11.0 Hz), 4.05-4.10 (1H, m), 4.08
(2H, s), 4.34 (1H, s), 4.39-4.44 (1H, m), 4.53 (1H, d, J=12.0 Hz),
4.65 (1H, d, J=12.0 Hz), 5.12 (1H, s), 5.19 (1H, dd, J=12.0, 3.0
Hz), 5.41 (1H, dd, J=16.0, 7.3 Hz), 5.50 (1H, ddd, J=12.3, 4.0, 3.0
Hz), 5.98 (1H, d, J=2.0 Hz), 6.08 (1H, d, J=16.0 Hz), 7.29-7.38
(5H, m); .sup.13C-NMR (125 MHz, CDCl.sub.3) .delta. 14.0, 15.3,
19.4, 20.0, 21.4, 22.5, 23.9, 24.7, 28.8, 29.0, 31.2, 31.6, 32.6,
34.5, 34.6, 37.1, 38.5, 40.8, 42.2, 43.2, 45.0, 51.1, 65.0, 65.8,
66.5, 71.0, 71.1, 71.1, 73.2, 74.0, 75.0, 75.5, 98.7, 101.2, 119.7,
126.9, 127.7, 127.8, 128.4, 138.2, 142.7, 151.4, 166.8, 167.2,
170.1, 170.1, 172.1; HRMS calc'd for C.sub.49H.sub.71ClO.sub.16
(+1Na): 973.4350; found: 973.4328;
[.alpha.].sup.25.0.sub.d:-3.04.degree. (c 1.34, CH.sub.2Cl.sub.2).
##STR74##
705.2 (Formula 705 where R is OH, R' is OBn, R.sup.3 is OH, R.sup.5
is .dbd.O, R.sup.8 is t-Bu-OH, R.sup.9 is H, R.sup.20 is
--O--CO--C.sub.7H.sub.15, R.sup.21 is .dbd.CH--CO.sub.2Me and
R.sup.26 is Me)
[0349] To 705.1 (7.4 mg, 7.785 .mu.mol) in THF (0.66 mL) was added
thiourea (66 mg, 0.84 mmol) and the resulting slurry was stirred at
room temperature for 3 days. The reaction was then added directly
to a silica column and eluted (50% to 58% to 70% EtOAc/pentane) to
afford 705.2 (a compound of Formula III where R.sup.3 is OH,
R.sup.5 is .dbd.O, R.sup.8 is t-Bu-OH, R.sup.9 is H, R.sup.20 is
--O--CO--C.sub.7H.sub.15, R.sup.21 is .dbd.CH--CO.sub.2Me and
R.sup.26 is Me). (6.3 mg, 92%). 705.2: R.sub.f (50%
EtOAc/pentane)=0.18; IR (film): 3423.1, 2926.7, 2856.8, 1726.4,
1659.2, 1376.1, 1253.2, 1159.3, 1098.3, 980.8, 799.1, 729.7
cm.sup.-1; .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 0.77 (3H, s),
0.86 (3H, t, J=10.8 Hz), 0.96 (3H, s), 1.03 (3H, s), 1.18 (3H, d,
J=8.1 Hz), 1.20 (3H, s), 1.22-1.34 (110H, m), 1.68-1.82 (3H, m),
2.01-2.12 (3H, m), 2.18-2.33 (3H, m), 2.45 (1H, dd, J=12.6, 4.5
Hz), 2.62-2.67 (2H, m), 2.85 (1H, dd, J=12.6, 3.6 Hz), 3.09-3.21
(2H, m), 3.60-3.88 (5H, m), 3.68 (3H, s), 3.93-4.00 (1H, m), 4.09
(1H, dd, J=11.1, 4.5 Hz), 4.35 (1H, s), 4.35-4.60 (1H, m), 4.52
(1H, d, J=12.0 Hz), 4.65 (1H, d, J=12.0 Hz), 5.06 (1H, d, J=9.6
Hz), 5.12 (1H, s), 5.18 (1H, d, J=7.2 Hz), 5.42 (1H, dd, J=15.9,
7.2 Hz), 5.44-5.51 (1H, m), 5.98 (1H, s), 6.08 (1H, d, J=15.9 Hz),
7.35-7.39 (5H, m); .sup.13C-NMR (125 MHz, CDCl.sub.3) .delta. 14.1,
15.3, 18.4, 19.4, 22.4, 22.5, 23.9, 24.7, 28.9, 29.0, 29.7, 31.2,
31.6, 32.7, 34.5, 34.7, 36.8, 39.7, 42.3, 43.2, 45.0, 51.1, 65.0,
65.9, 66.5, 69.0, 71.1, 71.2, 74.0, 75.0, 77.2, 98.7, 101.3, 119.7,
126.9, 127.7, 127.9, 128.4, 138.2, 142.8, 151.5, 166.9, 170.1,
171.8, 172.1; HRMS calc'd for C.sub.47H.sub.69O.sub.15 (+1Na):
897.4595; found: 897.4612; [.alpha.].sup.25.0.sub.d:-9.50.degree.
(c 0.47, CH.sub.2Cl.sub.2). ##STR75##
Formula 705.3
[0350] To myristic acid (10 mg, 0.044 mmol) in toluene (2.14 mL) at
rt under nitrogen is added triethylamine (23.3 .mu.L, 0.175 mmol)
followed by 2,4,6-trichlorobenzoylchloride (6.8 .mu.L, 0.044 mmol).
The resulting solution was stirred for 45 min. An aliquot of this
solution (63 .mu.L, 0.0013 mmol) was added to a solution of 705.2
(1 mg, 0.00114 mmol) and DMAP (0.7 mg, 0.0059 mmol) in toluene (500
.mu.L). The slightly yellow, cloudy solution is stirred at rt for
30 min. and then added directly to a silica column and eluted (30%
EtOAC/pentane). The eluted material is then re-chromatographed (30%
EtOAC/pentane). The resulting material was then dissolved in EtOAc
(500 .mu.L) and Pearlman's catalyst (2 mg) was added. The resulting
suspension was evacuated and re-filled with hydrogen (5 times)
while the reaction was stirred vigorously. After 30 min., the
reaction is added directly to a silica column and eluted (EtOAc).
This provided C7 myristate analogue 705.3 (360 .mu.g, 32%-2 steps)
(a compound of Formula III where R.sup.3 is OH, R.sup.5 is .dbd.O,
R.sup.8 is t-Bu-myristate, R.sup.9 is H, R.sup.20 is
--O--CO--C.sub.7H.sub.15, R.sup.21 is .dbd.CH--CO.sub.2Me and
R.sup.26 is Me). R.sub.f (40% EtOAc/pentane)=0.19. IR (film)
3256.8, 2915.8, 2845.2, 1746.4, 1722.9, 1158.4, 1029.0, 864.4,
793.8 cm.sup.-1. .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.
0.86-0.94 (12H, m), 1.05 (3H, s), 1.14-1.38 (38H, m), 1.59-1.63
(2H, m), 1.63-1.88 (3H, m), 2.03-2.07 (3H, m), 2.31-2.42 (4H, m),
2.47 (1H, dd, J=12.8, 3.8 Hz), 2.72-2.74 (2H, m), 2.86 (1H, dd,
J=12.5, 5.0 Hz), 3.70 (3H, s), 3.62-3.75 (3H, m), 3.78-3.87 (3H,
m), 3.99-4.04 (1H, m), 4.09-4.14 (1H, m), 4.37-4.50 (2H, m), 5.15
(1H, s), 5.16 (1H, d, J=7.4 Hz), 5.21 (1H, d, J=12.0 Hz), 5.33-5.34
(1H, m), 5.43 (1H, dd, J=15.6, 7.4 Hz), 6.01 (1H, s), 6.07 (1H, d,
J=15.6 Hz). .sup.13C-NMR (500 MHz, CDCl.sub.3) .delta. 14.1, 19.9,
20.5, 21.1, 22.7, 23.2, 23.9, 24.7, 24.7, 24.9, 26.7, 29.1, 29.3,
29.4, 29.4, 29.7, 30.2, 31.1, 31.6, 31.9, 33.5, 33.7, 34.3, 34.5,
35.8, 37.2, 38.4, 42.3, 43.3, 45.0, 51.1, 65.0, 65.9, 66.6, 69.4,
70.0, 73.3, 73.5, 74.0, 75.9, 77.2, 98.7, 101.2, 119.8, 126.9,
128.8, 142.7, 151.3, 166.8, 170.4, 170.9, 172.4. HRMS (FAB) calc'd
for C.sub.54H.sub.90O.sub.16Na: 1017.6133, found: 1017.6127.
[.alpha.].sup.21.1.sub.D=-7.14.degree. (c 0.035,
CH.sub.2Cl.sub.2).
3D. Formula V--Bryostatin Analogue Containing Diester Linker
(903.1)
[0351] ##STR76##
[0352] To a solution of silyl ether 111 from Example 1C (1.17 g,
1.44 mmol) and pyridine (2.07 mL, 25.65 mmol) in 12 mL THF in a
polypropylene vial was added HF/pyridine complex (0.83 mL, 28.83
mmol) at rt. The solution was stirred for 24 hours and diluted with
EtOAc. The organic layer was washed with sat. CuSO.sub.4 and brine,
dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the
corresponding alcohol (not shown) as a pale yellow oil. To the
alcohol (24.6 mg, 0.036 mmol) in methylene chloride (0.7 mL) was
added DMAP (24.5 mg, 0.201 mmol) followed by succinic anhydride
(8.6 mg, 0.086 mmol) at rt. The solution was heated to 42.degree.
C. for 3 hours and then slowly cooled to rt. Col. chromatography
(40% EtOAc+1% AcOH/hexane) provided crude 901.1 (28.6 mg, 0.0353
mmol). ##STR77##
902.1 (Formula 902 where R.sup.20 is --O--CO--C.sub.7H.sub.15,
R.sup.21 is .dbd.CH--CO.sub.2Me
[0353] To crude 901.1 (28.6 mg) in methylene chloride (0.8 mL) and
water (9.5 .mu.L) was added DDQ (10.4 mg, 0.046 mmol) at rt. After
stirring for 2 hours, the reaction was quenched with a saturated
aqueous solution of ammonium chloride and extracted with EtOAc
(3.times.5 mL). The combined organics were then dried over sodium
sulfate and the solvent was removed in vacuo. Chromatography (40%
EtOAc+1% AcOH/hexane) provided seco acid 902.1 (21.9 mg, 0.032
mmol, 91% in 2 steps). ##STR78##
[0354] To 902.1 (5 mg, 7.38 .mu.mol) in acetonitrile (0.4 mL) and
water (42 .mu.L) at rt was added 48% aqueous HF dropwise (24 .mu.L,
0.738 mmol). The reaction was stirred for 40 min. and then quenched
with a saturated aqueous solution of sodium bicarbonate (5 mL). The
layers were separated and then the aqueous layer was extracted with
EtOAc (6.times.5 mL). The combined organics were dried over sodium
sulfate and then the solvent was removed in vacuo. The resulting
clear oil (not shown) was then used immediately in the next
step.
[0355] To DMAP (9 mg, 0.074 mmol) and DMAP-HCl (8.2 mg, 0.052 mmol)
in methylene chloride (1.4 mL) was added DCC (10.6 mg, 0.052 mmol)
at rt. The clear oil from the preceding step in methylene chloride
(2.2 mL) was then added over 3 h. The resulting mixture was stirred
at rt for 4 h and then quenched with a saturated aqueous solution
of sodium bicarbonate (5 mL). The layers were separated and then
the aqueous layer was extracted with EtOAc (3.times.5 mL). The
combined organics were then dried over sodium sulfate and the
solvent was removed in vacuo. Silica gel chromatography (30%
EtOAc/hexane) provided corresponding crude macrocycle (not shown)
as a colorless oil.
[0356] The crude macrocycle from the preceding step was dissolved
in ethyl acetate (2.6 mL) and Pd(OH).sub.2/C (2.4 mg, 20% wt. on
carbon) was added. The resulting suspension was evacuated and
refilled with 1 Atm. hydrogen gas (.times.5) and was vigorously
stirred under a hydrogen atmosphere for 3 hours. The crude mixture
was pipetted directly onto a silica gel column and the product was
eluted (50% EtOAc/hexane) to afford of bryostatin analogue 903.1
(0.3 mg, 7%-3 steps) (a compound of Formula V where p is 2,
R.sup.20 is --O--CO--C.sub.7H.sub.15, R.sup.21 is
.dbd.CH--CO.sub.2Me and R.sup.26 is Me) as a white solid.
Example 4
Bryostatin Analogues Containing Selected C20 Ester Substituent
[0357] This example illustrates methods for preparing bryostatin
compounds and analogues that contain selected ester substituents at
C20.
4A. Acetyl C20 Ester (702.2)
[0358] ##STR79##
[0359] To a solution of enal 13, prepared as described for compound
13 in Wender et al. (1998a) (180 mg, 0.03 mmol) in 0.5 mL of MeOH
at rt was added pyridinium p-toluenesulfonate (PPTS, 2 mg,
catalytic) and trimethylorthoformate (5 drops). The progress of the
reaction was monitored by thin layer chromatography (TLC). After 30
min the reaction was quenched with 1.0 mL Et.sub.3N. The solvent
was removed under reduced pressure to afford the expected crude
dimethylacetal product (not shown). This product was immediately
dissolved in MeOH (0.5 mL) and K.sub.2CO.sub.3 (3 mg, catalytic).
The progress of the reaction was monitored by TLC. After 30 min the
reaction was quenched. The solution was quenched with sat.
NaHCO.sub.3, diluted with EtOAc (10 mL), washed with H.sub.2O,
dried over MgSO.sub.4 and concentrated under reduced pressure to
afford the crude C20 free hydroxyl product (not shown). The crude
product was immediately dissolved in methylene chloride (0.5 mL)
and acetic anhydride (0.3 mL, excess) and DMAP (5 mg, catalytic)
was added at rt. The progress of the reaction was monitored by TLC.
After 30 min the reaction was quenched. The solution was quenched
with sat. NaHCO.sub.3, diluted with EtOAc (10 mL), washed with
H.sub.2O, dried over MgSO.sub.4 and concentrated under reduced
pressure. The crude product was purified by column chromatography
on silica gel with 20% EtOAc-hexanes as eluant affording 14 mg (82%
for four steps) of dimethylacetal 37. R.sub.f(20% ethyl
acetate/hexanes)=0.17; R.sub.f (25% EtOAc/hexanes)=0.44; IR 2927,
2859, 1744, 1719, 1687, 1514, 1249, 1156, 1103, 1079, 1037
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.85 (6H, m)
1.16-1.30 (10H, m), 1.14 (3H, s), 1.18 (3H, s), 1.75 (1H, m),
1.98-2.17 (3H, m), 2.32 (1H, m), 3.27 (3H, s), 3.52 (1H, d, J=16.5
Hz), 3.68 (3H, s), 3.79 (3H, s), 3.87 (1H, m), 3.95 (1H, m), 4.09
(1H, m), 4.47 (2H, ABq, J=11.4 Hz), 5.41 (1H, s), 5.88 (1H, s),
5.91 (1H, dd, J=16.2, 7.7 Hz), 6.83 (2H, d, J=8.7 Hz), 7.16 (2H, d,
J=8.7 Hz), 7.27-7.35 (6H, m), 9.44 (1H, d, J=7.7 Hz, C15); .sup.13C
NMR (75 MHz, CDCl.sub.3) .delta. 14.1, 21.7, 22.6, 24.0, 24.6,
28.9, 29.0, 31.7, 32.6, 34.5, 36.2, 47.5, 51.3, 51.5, 55.4, 69.2,
71.3, 71.8, 74.4, 76.3, 102.7, 114.1, 118.2, 127.1, 127.7, 127.9,
128.7, 129.5, 130.7, 138.9, 151.6, 159.6, 166.6, 167.3, 172.1,
195.0; [.alpha.].sub.D.sup.20=-0.7.degree. (c 1.7,
CH.sub.2Cl.sub.2). ##STR80##
303.1 (Formula 303 where R.sup.20 is --O--CO-Me, R.sup.21 is
.dbd.CH--CO.sub.2Me)
[0360] To a solution of dimethylacetal 37 (14 mg, 0.02 mmol) in 0.6
mL 1% aqueous CH.sub.2Cl.sub.2 was added solid
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 10 mg, 0.03 mmol)
at rt. The mixture was stirred for 2 h, pipetted directly onto a
column of silica gel, and the product eluted with 35% EtOAc/hexanes
to provide intermediate alcohol 303.1 (10 mg, 91%) as a colorless
oil: R.sub.f (35% EtOAc/hexanes)=0.22; IR 3528, 2930, 2858, 1745,
1720, 1686, 1458, 1437, 1380 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 0.86 (3H, t, J=6.9 Hz), 1.13 (3H, s), 1.17 (3H,
s), 1.25 (10H, m), 1.52 (2H, m), 1.71 (2H, m), 2.12 (2H, m), 2.35
(1H, t, J=14.1 Hz), 2.60 (1H, d, J=3.6 Hz), 3.41 (3H, s, C19
OCH.sub.3), 3.45 (1H, m), 3.68 (3H, s), 3.82 (1H, s), 4.24 (1H, m),
4.55 (2H, ABq, J=11.4 Hz), 5.47 (1H, s), 5.86 (1H, s), 5.91 (1H,
dd, J=15.9, 7.5 Hz), 7.29 (1H, d, J=15.9 Hz), 7.34 (5H, s), 9.52
(1H, d, J=7.5 Hz); .sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. 13.6,
15.3, 21.6, 22.4, 23.5, 24.7, 28.6, 28.8, 31.5, 32.8, 34.1, 39.5,
47.4, 51.1, 51.2, 68.3, 70.9, 71.0, 71.1, 78.8, 102.4, 117.4,
126.8, 127.9, 128.0, 128.5, 138.1, 151.8, 166.4, 167.3, 171.4,
194.5; [.alpha.].sub.D.sup.20=-21.0.degree. (c 1.0,
CH.sub.2Cl.sub.2). ##STR81##
304.1 (Formula 304 where R.sup.20 is --O--CO-Me, R.sup.21 is
.dbd.CH--CO.sub.2Me)
[0361] Alcohol 303.1 (10 mg, 0.02 mmol) was dissolved in 1.1 mL
CH.sub.3CN/H.sub.2O (9:1) and treated with 48% aqueous HF (200
.mu.L, 300 mol % excess) at rt. The resulting mixture was stirred
for 1 h, quenched with sat. NaHCO.sub.3 and diluted with 10 mL
EtOAc. The aqueous layer was separated and extracted with EtOAc
(2.times.). The combined organics were dried over Na.sub.2SO.sub.4
and concentrated in vacuo to afford crude hemiketal enal 304.1 as a
colorless oil. The crude product was purified by column
chromatography on silica gel with 35% EtOAc-hexanes as eluant
affording 8 mg (89%) of hemiketal enal 304.1. R.sub.f (35%
EtOAc/hexanes)=0.15; IR 3528, 2930, 2858, 1745, 1720, 1686, 1458,
1437, 1380 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
0.86 (3H, t, J=6.9 Hz, octanoate Me), 1.13 (3H, s, C18 Me), 1.17
(3H, s, C18 Me), 1.25 (10H, m), 1.52 (2H, m), 1.71 (2H, m), 2.12
(2H, m), 2.35 (1H, t, J=14.1 Hz), 2.60 (1H, d, J=3.6 Hz), 3.41 (3H,
s, C19 OCH.sub.3), 3.45 (1H, m), 3.68 (3H, s, methyl ester), 3.82
(1H, s), 4.24 (1H, m), 4.44 (1H, d, J=11.1 Hz, CH.sub.2Ph), 4.69
(1H, d, J=11.4 Hz, CH.sub.2Ph), 5.47 (1H, s, C20), 5.86 (1H, s,
C34), 5.91 (1H, dd, J=15.9, 7.5 Hz, C16), 7.29 (1H, d, J=15.9 Hz,
C17), 7.34 (5H, s, Ph), 9.52 (1H, d, J=7.5 Hz, C15); .sup.13C-NMR
(75 MHz, CDCl.sub.3) .delta. 13.8, 15.4, 21.7, 22.3, 23.6, 24.3,
28.7, 28.8, 31.4, 32.7, 34.2, 39.5, 47.3, 51.1, 51.2, 68.3, 70.8,
71.0, 71.1, 78.1, 102.3, 117.4, 126.8, 128.0, 128.1, 128.6, 138.1,
151.8, 166.4, 167.1, 171.7, 194.7;
[.alpha.].sub.D.sup.20=-19.0.degree. (c 1.4, CH.sub.2Cl.sub.2).
##STR82##
701.1 (Formula 701 where R is OH, R' is OBn, R.sup.3 is TBSO,
R.sup.20 is --O--CO-Me, R.sup.21 is .dbd.CH--CO.sub.2Me, R.sup.26
is Me and X is Oxygen)
[0362] Carboxylic acid 407 (Example 2B, 15 mg, 0.03 mmol) and
Et.sub.3N (16.5 .mu.L, 0.12 mmol) were dissolved in 300 .mu.L
toluene and treated with 2,4,6-trichlorobenzoylchloride (4.8 .mu.L,
0.03 mmol) dropwise at rt. After 1 h at rt, a toluene solution of
freshly prepared 304.1 and 4-dimethylaminopyridine (14 mg, 0.12
mmol) was added gradually and stirring was continued for 40 min.
The crude mixture was pipetted directly onto a column of silica gel
and the product eluted with 20% EtOAc/hexanes to provide ester
701.1 as a colorless oil (15 mg, 63%). R.sub.f (35%
EtOAc/hexanes)=0.71; IR 3487, 2927, 2856, 1723, 1689, 1455, 1379,
1228, 1156, 1113, 1084, 1032, 981 cm.sup.-1; .sup.1H NMR (300 MHz,
C6D.sub.6) .delta. 0.80 (1H, t, J=12.7 Hz), 0.93 (3H, t, J=7.0 Hz),
0.97 (3H, d, J=6.6 Hz), 1.10-1.36 (24H, m), 1.36-1.85 (21H, m),
1.91-2.13 (6H, m), 2.39 (1H, t, J=12.8 Hz), 2.79 (1H, d, J=13.2
Hz), 2.93 (1H, m), 3.05 (1H, m), 3.29 (3H, s), 3.33 (1H, s),
3.37-3.48 (2H, m), 3.80 (1H, dd, J=11.4, 5.1 Hz), 3.95-4.05 (2H,
m), 4.15 (1H, td, J=10.8, 0.9 Hz), 4.23 (1H, d, J=13.5 Hz), 4.40
(1H, d, J=12.0 Hz), 4.47 (1H, d, J=12.0 Hz), 5.57 (1H, s), 5.64
(1H, dd, J=10.6, 4.6 Hz), 6.05 (1H, dd, J=16.1, 7.6 Hz), 6.39 (1H,
s), 7.10-7.35 (5H, m), 7.45 (1H, d, J=16.1 Hz), 9.60 (1H, d, J=7.6
Hz); .sup.13C-NMR (75 MHz, C.sub.6D.sub.6) .delta. 14.1, 15.1,
19.3, 20.1, 21.3, 22.3, 22.4, 22.7, 23.8, 24.0, 24.6, 24.6, 29.0,
29.0, 29.3, 31.3, 31.6, 31.8, 34.2, 34.5, 35.2, 35.7, 36.2, 37.6,
43.5, 45.7, 51.6, 59.1, 64.9, 66.7, 71.1, 72.0, 72.9, 74.1, 75.3,
77.1, 100.2, 100.6, 121.2, 127.2, 127.3, 127.9, 128.6, 138.9,
151.2, 164.5, 166.3, 171.5, 175.1, 193.4;
[.alpha.].sup.20.sub.D-19.degree. (c 1.5, CH.sub.2Cl.sub.2).
##STR83##
701.2 (Formula 701 where R is OH, R' is OBn, R.sup.3 is OH.
R.sup.20 is --O--CO-Me R.sup.21 is .dbd.CH--CO.sub.2Me, R.sup.26 is
Me and X is Oxygen)
[0363] To ester 701.1 (13 mg, 0.02 mmol) in THF (0.5 mL) was added
pyridine (360 .mu.L, 0.45 mmol) followed by 70% HF/pyridine (144
.mu.L, 500 mol % excess) and stirred for 20 hours. The reaction was
then quenched with a saturated solution of sodium bicarbonate. The
biphasic mixture was extracted with ethyl acetate (.times.4) and
the combined organics were dried over sodium sulfate. The solvent
was removed in vacuo to provide crude C3 hydroxyester 701.2. The
crude mixture was pipetted directly onto a column of silica gel and
the product eluted with 35% EtOAc/hexanes to provide ester 701.2 as
a colorless oil (9 mg, 82%). R.sub.f (40% EtOAc/hexanes)=0.19; IR
3522, 2927, 2857, 1724, 1664, 1230, 1158, 1136, 1107, 979
cm.sup.-1; .sup.1H NMR (400 MHz, C.sub.6D.sub.6) .delta. 0.84 (3H,
t, J=5.4 Hz), 0.88-0.96 (5H, m), 1.00 (3H, d, J=4.8 Hz), 1.02-1.55
(27H, m), 1.63-1.81 (2H, m), 1.82-1.94 (2H, m), 2.03 (1H, br t,
J=5.2 Hz), 2.19-2.27 (1H, m), 2.34 (1H, dt, J=9, 1.5 Hz), 2.94-3.01
(2H, m), 3.22 (1H, s), 3.58 (1H, br d, J=3.6 Hz), 3.68-3.74 (1H,
m), 3.84-3.88 (1H, m), 3.94 (1H, dd, J=8.6, 3.1 Hz), 4.23 (1H, dd,
J=10.4, 1.7 Hz), 4.31 (1H, br t, J=8.1 Hz), 5.36-5.41 (1H, m), 5.50
(1H, s), 5.61 (1H, d, J=5.4 Hz), 6.00 (1H, dd, J=12.0, 5.4 Hz),
6.36 (1H, s), 6.53 (1H, d, J=12.0 Hz); .sup.13C NMR (100 MHz,
C.sub.6D.sub.6) .delta. 14.2, 19.4, 19.8, 22.4, 22.9, 24.0, 24.5,
25.0, 29.1, 29.2, 30.2, 31.8, 31.9, 32.0, 33.1, 34.6, 34.9, 35.4,
36.5, 43.6, 45.6, 50.7, 66.1, 66.7, 69.6, 73.2, 74.7, 75.8, 76.3,
77.5, 98.7, 102.6, 120.5, 140.1, 151.2, 151.5, 166.5, 171.5,
174.2[.alpha.].sup.20.sub.D -13.5.degree. (c 0.9, CDCl.sub.3).
##STR84##
702.2 (Formula 702 where R.sup.3 is OH, R.sup.20 is --O--CO-Me
R.sup.21 is .dbd.CH--CO.sub.2Me, R.sup.26 is Me and X is
Oxygen)
[0364] To a solution of C3 hydroxyester 701.2 (8 mg, 0.01 mmol) in
2.0 mL CH.sub.2Cl.sub.2 was added 4 .ANG. molecular sieves and the
mixture was aged for 20 min. 45-50 beads of Amberlyst-15 sulfonic
acid resin were added and the mixture was stirred at rt for 2 h.
The crude mixture was pipetted directly onto a column of silica gel
and the product eluted with 35% EtOAc/hexanes to provide the
expected macrocyclic product (not shown) as a colorless oil (5 mg,
83%). R.sub.f (35% EtOAc/hexanes)=0.21; IR 3522, 2927, 2857, 1724,
1664, 1230, 1158, 1136, 1107, 979 cm.sup.-1; .sup.1H NMR (400 MHz,
C.sub.6D.sub.6) .delta. 0.84 (3H, t, J=5.4 Hz), 0.88-0.96 (5H, m),
1.00 (3H, d, J=4.8 Hz), 1.02-1.55 (27H, m), 1.63-1.81 (2H, m),
1.82-1.94 (2H, m), 2.03 (1H, br t, J=5.2 Hz), 2.19-2.27 (1H, m),
2.34 (1H, dt, J=9, 1.5 Hz), 2.94-3.01 (2H, m), 3.22 (1H, s), 3.58
(1H, br d, J=3.6 Hz), 3.68-3.74 (1H, m), 3.84-3.88 (1H, m), 3.94
(1H, dd, J=8.6, 3.1 Hz), 4.23 (1H, dd; J=10.4, 1.7 Hz), 4.31 (1H,
br t, J=8.1 Hz), 5.36-5.41 (1H, m), 5.50 (1H, s), 5.61 (1H, d,
J=5.4 Hz), 6.00 (1H, dd, J=12.0, 5.4 Hz), 6.36 (1H, s), 6.53 (1H,
d, J=12.0 Hz); .sup.13C NMR (100 MHz, C.sub.6D.sub.6) .delta. 14.2,
19.4, 19.8, 22.4, 22.9, 24.0, 24.5, 25.0, 29.1, 29.2, 30.2, 31.8,
31.9, 32.0, 33.1, 34.6, 34.9, 35.4, 36.5, 43.6, 45.6, 50.7, 66.1,
66.7, 69.6, 73.2, 74.7, 75.8, 76.3, 77.5, 98.7, 102.6, 120.5,
140.1, 151.2, 151.5, 166.5, 171.5, 174.2.
[0365] The macrocyclic product from the preceding step was
dissolved in 0.5 mL EtOAc and 2.2 mg Pd(OH).sub.2 (20% wt. on
carbon) was added. The resulting suspension was vigorously stirred
under balloon pressure of hydrogen gas for 35 min. The crude
mixture was pipetted directly onto a column of silica gel and the
product was eluted with 60% EtOAc/hexanes to afford acetate
analogue 702.2 (4 mg, 93%) (a compound of Formula II where R.sup.3
is OH, R.sup.20 is --O--CO-Me, R.sup.21 is .dbd.CH--CO.sub.2Me,
R.sup.26 is Me and X is oxygen) as a white semi-solid. R.sub.f (50%
EtOAc/hexanes)=0.16; IR 3522, 2927, 2857, 1724, 1664, 1230 IR
(neat)=3455, 3319, 2929, 2856, 1735, 1380, 1230, 1138, 976
cm.sup.-1; .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.37 (3H, br.
s), 0.77-0.92 (14H, m), 1.06 (3H, d, J=6.4 Hz), 1.07 (1H, t, J=11.0
Hz), 1.10-1.28 (5H, m), 1.27 (3H, s), 1.50 (3H, s), 1.57-1.78 (4H,
m), 2.03 (2H, t, J=7.4 Hz), 2.17 (1H, dd, J=9.9, 0.5 Hz), 2.37 (1H,
m), 2.40 (1H, m), 2.85 (1H, t, J=11.2 Hz), 2.96 (1H, t, J=10.8 Hz),
3.15 (3H, s), 3.68-3.75 (3H, m), 3.91 (1H, dd, J=11.2, 4.0 Hz),
4.12 (1H, t, J=9.7 Hz), 4.35 (1H, dd, J=13.9, 2.3 Hz), 4.48 (1H,
td, J=11.0, 2.8 Hz), 4.70 (1H, d, J=12.1 Hz), 5.44 (1H, quint.,
J=4.8 Hz), 5.54 (1H, d, J=7.3 Hz), 5.69 (1H, s), 5.76 (1H, s), 5.85
(1H, dd, J=16.1, 7.5 Hz), 6.40 (1H, d, J=1.8 Hz), 6.50 (1H, d,
J=15.9 Hz); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 14.3, 19.6,
12.0, 22.9, 23.2, 24.9, 25.0, 29.2, 29.2, 31.4, 31.5, 31.9, 32.9,
34.7, 36.3, 39.9, 42.6, 43.2, 45.4, 50.5, 65.2, 66.2, 67.0, 70.4,
74.2, 74.7, 75.3, 75.9, 78.5, 99.7, 103.1, 120.5, 142.5, 152.5,
171.6, 172.5; [.alpha.].sup.25.sub.D=-9.0.degree. (c=0.36,
CDCl.sub.3).
4B. Heptanoate C20 Ester (702.3)
[0366] ##STR85##
308.1 (Formula 308 where R.sup.20a is Heptenoate, and R.sup.21 is
.dbd.CH--CO.sub.2Me)
[0367] To solution of the enal of Formula 305 (in which R.sup.20 is
C.sub.7H.sub.15), prepared as described for compound 13 in Wender
et al. (1998a), (224 mg, 0.04 mmol) in 0.5 mL of MeOH at rt was
added PPTS (2 mg, catalytic) and trimethylorthoformate (1 drop).
The progress of the reaction was monitored by TLC. After 30 min the
reaction was quenched with 1.0 mL Et.sub.3N. The solvent was
removed under reduced pressure to afford the corresponding crude
dimethylacetal product of Formula 306. This product was immediately
dissolved in MeOH (2.0 mL) and K.sub.2CO.sub.3 (3 mg, catalytic).
The progress of the reaction was monitored by TLC. After 30 min the
reaction was quenched. The solution was quenched with sat.
NaHCO.sub.3, diluted with EtOAc (10 mL), washed with H.sub.2O,
dried over MgSO.sub.4 and concentrated under reduced pressure to
afford the corresponding crude C20 free hydroxyl product of Formula
307.
[0368] Heptenoic acid (6 mg, 0.05 mmol) and Et.sub.3N (21 .mu.L,
0.16 mmol) were dissolved in 600 .mu.L toluene and treated with
2,4,6-trichlorobenzoylchloride (7.0 .mu.L, 0.05 mmol) dropwise at
rt. After 1 h at rt, a toluene solution of the freshly prepared C20
free hydroxyl product of Formula 307 and 4-dimethylaminopyridine
(DMAP, 20 mg, 0.17 mmol) was added gradually and stirring was
continued for 40 min. After 30 min the reaction was quenched. The
solution was quenched with sat. NaHCO.sub.3, diluted with EtOAc (10
mL), washed with H.sub.2O, dried over MgSO.sub.4 and concentrated
under reduced pressure. The crude product was purified by column
chromatography on silica gel with 20% EtOAc-hexanes as eluant
affording 21 mg (84%) of the corresponding dimethylacetal, C20
heptenoate product of Formula 308.1. R.sub.f (20% ethyl
acetate/hexanes)=0.22; IR 2927, 2859, 1744, 1719, 1687, 1514, 1249,
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.85 (6H, m)
1.16-1.30 (10H, m), 1.14 (3H, s), 1.18 (3H, s), 1.75 (1H, m),
1.98-2.17 (3H, m), 2.32 (1H, m), 3.27 (3H, s), 3.52 (1H, d, J=16.5
Hz), 3.68 (3H, s), 3.79 (3H, s), 3.87 (1H, m), 3.95 (1H, m), 4.09
(1H, m), 4.42 (2H, ABq, J=11.0 Hz), 4.59 (1H, d, J=11.0 Hz), 4.65
(1H, d, J=11.8 Hz), 4.98 (1H, s), 5.02 (1H, d, J=15.2 Hz), 5.41
(1H, s), 5.88 (1H, s), 5.91 (1H, dd, J=16.2, 7.7 Hz), 6.83 (2H, d,
J=8.7 Hz), 7.16 (2H, d, J=8.7 Hz), 7.27-7.35 (6H, m), 9.44 (1H, d,
J=7.7 Hz); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.1, 21.7,
22.6, 24.0, 24.6, 28.9, 29.0, 31.7, 32.6, 34.5, 36.2, 47.5, 51.3,
51.5, 55.4, 69.2, 71.3, 71.8, 74.4, 76.3, 102.7, 114.1, 118.2,
127.1, 127.7, 127.9, 128.7, 129.5, 130.7, 138.9, 151.6, 159.6,
166.6, 167.3, 172.1, 195.0.
[0369] To a solution of 308.1 (17 mg, 0.02 mmol) in 0.5 mL 1%
aqueous CH.sub.2Cl.sub.2 was added solid
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 8 mg, 0.03 mmol) at
rt. The mixture was stirred for 2 h, pipetted directly onto a
column of silica gel, and the product eluted with 35% EtOAc/hexanes
to provide the corresponding intermediate alcohol (11 mg, 85%) as a
colorless oil. R.sub.f (50% EtOAc/hexanes)=0.55; IR 3528, 2930,
2858, 1745, 1720, 1686 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 0.86 (3H, t, J=6.9 Hz), 1.13 (3H, s), 1.17 (3H, s), 1.25
(10H, m), 1.52 (2H, m), 1.71 (2H, m), 2.12 (2H, m), 2.35 (1H, t,
J=14.1 Hz), 2.60 (1H, d, J=3.6 Hz), 3.41 (3H, s), 3.45 (1H, m),
3.68 (3H, s), 3.82 (1H, s), 4.24 (1H, m), 4.54 (2H, ABq, J=11.2
Hz), 5.47 (1H, s), 4.98 (1H, s), 5.02 (1H, d, J=15.2 Hz), 5.86 (1H,
s), 5.91 (1H, dd, J=15.9, 7.5 Hz), 7.29 (1H, d, J=15.9 Hz), 7.34
(5H, s), 9.52 (1H, d, J=7.5 Hz); .sup.13C-NMR (75 MHz, CDCl.sub.3)
.delta. 13.8, 15.4, 21.7, 22.4, 23.6, 24.4, 28.8, 28.8, 31.5, 32.8,
34.4, 39.5, 47.4, 51.1, 51.2, 68.3, 70.8, 71.1, 71.2, 78.3, 102.5,
117.3, 127.0, 127.9, 128.0, 128.5, 138.1, 151.7, 166.5, 167.4,
171.8, 194.6.
[0370] The intermediate alcohol (11 mg, 0.02 mmol) was dissolved in
1.0 mL CH.sub.3CN/H.sub.2O (9:1) and treated with 48% aqueous HF
(200 .mu.L, 300 mol % excess) at rt. The resulting mixture was
stirred for 1 h, quenched with sat. NaHCO.sub.3 and diluted with 10
mL EtOAc. The aqueous layer was separated and extracted with EtOAc
(2.times.). The combined organics were dried over Na.sub.2SO.sub.4
and concentrated in vacuo to afford the corresponding crude
hemiketal enal of Formula 303a as a colorless oil. The crude
product was purified by column chromatography on silica gel with
50% EtOAc-hexanes as eluant affording 8 mg (80%) of the C20
heptenoate hemiketal enal. R.sub.f (35% EtOAc/hexanes)=0.05; IR
3528, 2930, 2858, 1745, 1720, 1686, 1458, 1437, 1380 cm.sup.-1;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.86 (3H, t, J=6.9 Hz),
1.13 (3H, s), 1.17 (3H, s), 1.25 (10H, m), 1.52 (2H, m), 1.71 (2H,
m), 2.12 (2H, m), 2.35 (1H, t, J=14.1 Hz), 2.60 (1H, d, J=3.6 Hz),
3.41 (3H, s), 3.45 (1H, m), 3.68 (3H, s), 3.82 (1H, s), 4.24 (1H,
m), 4.52 (2H, ABq, J=11.4 Hz), 4.98 (1H, s), 5.02 (1H, d, J=15.2
Hz), 5.47 (1H, s), 5.86 (1H, s), 5.91 (1H, dd, J=15.9, 7.5 Hz),
7.29 (1H, d, J=15.9 Hz), 7.34 (5H, s), 9.52 (1H, d, J=7.5 Hz);
.sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. 13.9, 15.4, 21.7, 22.4,
23.6, 24.4, 28.8, 28.8, 31.4, 32.7, 34.2, 39.5, 47.4, 51.1, 51.2,
68.3, 70.9, 71.1, 71.1, 78.5, 102.4, 117.4, 126.8, 128.0, 128.0,
128.6, 138.1, 151.8, 166.4, 167.1, 171.8, 194.7.
[0371] Carboxylic acid 407 (21 mg, 0.04 mmol) and Et.sub.3N (19
.mu.L, 0.12 mmol) were dissolved in 400 .mu.L toluene and treated
with 2,4,6-trichlorobenzoylchloride (6.0 .mu.L, 0.04 mmol) dropwise
at rt. After 1 h at rt, a toluene solution of freshly prepared C20
heptenoate hemiketal enal (16 mg, 0.03 mmol) and
4-dimethylaminopyridine (17 mg, 0.13 mmol) was added gradually and
stirring was continued for 40 min. The crude mixture was pipetted
directly onto a column of silica gel and the product eluted with
20% EtOAc/hexanes to provide the expected ester (Formula 701 where
R is OH, R' is OBn, R.sup.3 is TBSO, R.sup.20 is heptenoate,
R.sup.21 is .dbd.CH--CO.sub.2Me and R.sup.26 is methyl) as a
colorless oil (24 mg, 80%). IR 3487, 2927, 2856, 1723, 1689, 1455,
1379 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.80 (1H,
t, J=12.7 Hz), 0.93 (3H, t, J=7.0 Hz), 0.97 (3H, d, J=6.6 Hz),
1.10-1.36 (24H, m), 1.36-1.85 (21H, m), 1.91-2.13 (6H, m), 2.39
(1H, t, J=12.8 Hz), 2.79 (1H, d, J=13.2 Hz), 2.93 (1H, m), 3.05
(1H, m), 3.29 (3H, s), 3.33 (1H, s), 3.37-3.48 (2H, m), 3.80 (1H,
dd, J=11.4, 5.1 Hz), 3.95-4.05 (2H, m), 4.15 (1H, td, J=10.8, 0.9
Hz), 4.23 (1H, d, J=13.5 Hz), 4.50 (2H, ABq, J=12.0 Hz), 4.98 (1H,
s), 5.00 (1H, d, J=15.2 Hz), 5.57 (1H, s), 5.64 (1H, dd, J=10.6,
4.6 Hz), 6.05 (1H, dd, J=16.1, 7.6 Hz), 6.39 (1H, s), 7.10-7.35
(5H, m), 7.45 (1H, d, J=16.1 Hz), 9.60 (1H, d, J=7.6 Hz);
.sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. 14.1, 15.1, 19.3, 20.1,
21.3, 22.3, 22.4, 22.7, 23.8, 24.0, 24.6, 24.6, 29.0, 29.0, 29.3,
31.3, 31.6, 31.8, 34.2, 34.5, 35.2, 35.7, 36.2, 37.6, 43.5, 45.7,
51.6, 59.1, 64.9, 66.7, 71.1, 72.0, 72.9, 74.1, 75.3, 77.1, 100.2,
100.6, 121.2, 127.2, 127.3, 127.9, 128.6, 138.9, 151.2, 164.5,
166.3, 171.5, 175.1, 193.4; [.alpha.].sup.20.sub.D -19.degree. (c
1.5, CH.sub.2Cl.sub.2).
[0372] To the ester prepared in the preceding step (21 mg, 0.03
mmol) in THF (0.5 mL) was added pyridine (360 .mu.L, 0.45 mmol)
followed by 70% HF/pyridine (144 .mu.L, 500 mol % excess) and
stirred for 20 hours. The reaction was then quenched with a
saturated solution of sodium bicarbonate. The biphasic mixture was
extracted with ethyl acetate (.times.4) and the combined organics
were dried over sodium sulfate. The solvent was removed in vacuo to
provide the corresponding crude C3 hydroxyester. The crude mixture
was pipetted directly onto a column of silica gel and the product
eluted with 30% EtOAc/hexanes to provide this corresponding ester
(where R.sup.3 is OH) as a colorless oil (13 mg, 68%). R.sub.f (30%
EtOAc/hexanes)=0.23; IR 3522, 2927, 2857, 1724, 1664, 1230, 1158,
1136, 1107, 979 cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 0.84 (3H, t, J=5.4 Hz), 0.88-0.96 (5H, m), 1.00 (3H, d,
J=4.8 Hz), 1.02-1.55 (27H, m), 1.63-1.81 (2H, m), 1.82-1.94 (2H,
m), 2.03 (1H, br t, J=5.2 Hz), 2.19-2.27 (1H, m), 2.34 (1H, dt,
J=9, 1.5 Hz), 2.94-3.01 (2H, m), 3.22 (1H, s), 3.58 (1H, br d,
J=3.6 Hz), 3.68-3.74 (1H, m), 3.84-3.88 (1H, m), 3.94 (1H, dd,
J=8.6, 3.1 Hz), 4.23 (1H, dd, J=10.4, 1.7 Hz), 4.31 (1H, br t,
J=8.1 Hz), 4.97 (1H, s), 5.02 (1H, d, J=15.2 Hz), 5.36-5.41 (1H,
m), 5.50 (1H, s), 5.61 (1H, d, J=5.4 Hz), 6.00 (1H, dd, J=12.0, 5.4
Hz), 6.36 (1H, s), 6.53 (1H, d, J=12.0 Hz); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 14.2, 19.4, 19.8, 22.4, 22.9, 24.0, 24.5, 25.0,
29.1, 29.2, 30.2, 31.8, 31.9, 32.0, 33.1, 34.6, 34.9, 35.4, 36.5,
43.6, 45.6, 50.7, 66.1, 66.7, 69.6, 73.2, 74.7, 75.8, 76.3, 77.5,
98.7, 102.6, 120.5, 140.1, 151.2, 151.5, 166.5, 171.5, 174.2;
[.alpha.].sup.20.sub.D -13.5.degree. (c 0.9, CDCl.sub.3).
[0373] To a solution of the C3 hydroxy ester of the preceding step
(12 mg, 0.01 mmol) in 1.0 mL CH.sub.2Cl.sub.2 was added 4 .ANG.
molecular sieves and the mixture was allowed to stand for 20 min.
45-50 beads of Amberlyst-15 sulfonic acid resin were added and the
mixture was stirred at rt for 2 h. The crude mixture was pipetted
directly onto a column of silica gel and the product eluted with
35% EtOAc/hexanes to provide the corresponding heptenoate
macrocycle as a colorless oil (7 mg, 70%). R.sub.f (35%
EtOAc/hexanes)=0.21; IR 3522, 2927, 2857, 1724, 1664, 1230, 1158,
1136, 1107, 979 cm.sup.-1; .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 0.84 (3H, t, J=5.4 Hz), 0.88-0.96 (5H, m), 1.00 (3H, d,
J=4.8 Hz), 1.02-1.55 (27H, m), 1.63-1.81 (2H, m), 1.82-1.94 (2H,
m), 2.03 (1H, br t, J=5.2 Hz), 2.19-2.27 (1H, m), 2.34 (1H, dt,
J=9, 1.5 Hz), 2.94-3.01 (2H, m), 3.22 (1H, s), 3.58 (1H, br d,
J=3.6 Hz), 3.68-3.74 (1H, m), 3.84-3.88 (1H, m), 3.94 (1H, dd,
J=8.6, 3.1 Hz), 4.23 (1H, dd, J=10.4, 1.7 Hz), 4.31 (1H, br t,
J=8.1 Hz), 4.99 (1H, s), 5.03 (1H, d, J=15.2 Hz), 5.36-5.41 (1H,
m), 5.50 (1H, s), 5.61 (1H, d, J=5.4 Hz), 6.00 (1H, dd, J=12.0, 5.4
Hz), 6.36 (1H, s), 6.53 (1H, d, J=12.0 Hz); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 14.2, 19.4, 19.8, 22.4, 22.9, 24.0, 24.5, 25.0,
29.1, 29.2, 30.2, 31.8, 31.9, 32.0, 33.1, 34.6, 34.9, 35.4, 36.5,
43.6, 45.6, 50.7, 66.1, 66.7, 69.6, 73.2, 74.7, 75.8, 76.3, 77.5,
98.7, 102.6, 120.5, 140.1, 151.2, 151.5, 166.5, 171.5, 174.2.
[0374] The crude macrocycle of the preceding step (2 mg, 0.01 mmol)
was dissolved in 0.5 mL EtOAc and 2.2 mg Pd(OH).sub.2 (20% wt. on
carbon) was added. The resulting suspension was vigorously stirred
under balloon pressure of hydrogen gas for 35 min. The crude
mixture was pipetted directly onto a column of silica gel and the
product was eluted with 60% EtOAc/hexanes to afford heptanoate
analogue (702.3) (Formula II where R.sup.3 is OH, R.sup.20 is
--O--CO--C.sub.6H.sub.13, R.sup.21 is .dbd.CH--CO.sub.2Me, R.sup.26
is methyl and X is oxygen) (1 mg, 63%) as a white semi-solid.
R.sub.f(50% EtOAc/hexanes)=0.21; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 0.37 (3H, br. s), 0.79-0.92 (14H, m), 1.06 (3H, d, J=6.4
Hz), 1.07 (1H, t, J=11.0 Hz), 1.10-1.25 (5H, m), 1.27 (3H, s), 1.50
(3H, s), 1.57-1.78 (4H, m), 2.03 (2H, t, J=7.4 Hz), 2.17 (1H, dd,
J=9.9/0.5 Hz), 2.37 (1H, m), 2.40 (1H, m), 2.85 (1H, t, J=11.2 Hz),
2.96 (1H, t, J=10.8 Hz), 3.15 (31H, s), 3.68-3.72 (3H, m), 3.91
(1H, dd, J=11.2, 4.0 Hz), 4.13 (1H, t, J=9.7 Hz), 4.35 (1H, dd,
J=13.9, 2.2 Hz), 4.48 (1H, td, J=11.0/2.8 Hz), 4.70 (1H, d, J=12.1
Hz), 5.44 (1H, quint., J=4.8 Hz), 5.54 (1H, d, J=7.3 Hz), 5.69 (1H,
s), 5.76 (1H, s), 5.85 (1H, dd, J=16.1, 7.5 Hz), 6.40 (1H, d, J=1.8
Hz), 6.50 (1H, d, J=15.9 Hz); .sup.13C NMR (125 MHz, CDCl.sub.3)
.delta. 14.2, 19.4, 19.8, 22.4, 22.9, 24.0, 24.5, 25.0, 29.1, 29.2,
30.2, 31.8, 31.9, 32.0, 33.1, 34.6, 34.9, 35.4, 36.5, 43.6, 45.6,
50.7, 66.1, 66.7, 69.6, 73.2, 74.7, 75.8, 76.3, 77.5, 98.7, 102.6,
120.5, 140.1, 151.2, 151.5, 166.5, 171.5, 174.2.
4C. Myristate C20 Ester (702.4)
[0375] To solution of the enal of Formula 305 (in which R.sup.20 is
C.sub.7H.sub.15) (180 mg, 0.03 mmol), prepared as described for
compound 13 in Wender et al. (1998a), in 0.5 mL of MeOH at rt was
added PPTS (2 mg, catalytic) and trimethylorthoformate (5 drops).
The progress of the reaction was monitored by TLC. After 30 min the
reaction was quenched with 1.0 mL Et.sub.3N. The solvent was
removed under reduced pressure to afford the corresponding crude
dimethylacetal according to Formula 306. The dimethylacetyl was
immediately dissolved in MeOH (0.5 mL) and K.sub.2CO.sub.3 (3 mg,
catalytic). The progress of the reaction was monitored by TLC.
After 30 min the reaction was quenched. The solution was quenched
with sat. NaHCO.sub.3, diluted with EtOAc (10 mL), washed with
H.sub.2O, dried over MgSO.sub.4 and concentrated under reduced
pressure to afford crude C20 free hydroxyl product of Formula 307,
which was reacted with myristic acid in the same manner as the
reaction of heptenoic acid in Example 4B. After 30 min the reaction
was quenched. The solution was quenched with sat. NaHCO.sub.3,
diluted with EtOAc (10 mL), washed with H.sub.2O, dried over
MgSO.sub.4 and concentrated under reduced pressure. The crude
product was purified by column chromatography on silica gel with
35% EtOAc-hexanes as eluant affording 14 mg (70% for three steps)
of the desired dimethylacetal myristate of Formula 308. Rf (20%
ethyl acetate/hexanes)=0.5; R.sub.f (35% EtOAc/hexanes)=0.50; IR
2927, 2859, 1744, 1719, 1687, 1514, 1249, 1156, 1103, 1079, 1037
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.0.85 (6H, m)
1.16-1.30 (10H, m), 1.14 (3H, s), 1.18 (3H, s), 1.75 (1H, m),
1.95-2.17 (3H, m), 2.32 (1H, m), 3.37 (3H, s), 3.52 (1H, d, J=16.5
Hz), 3.68 (3H, s), 3.79 (3H, s), 3.87 (1H, m), 3.95 (1H, m), 4.09
(1H, m), 4.55 (2H, ABq, J=11.0 Hz), 5.41 (1H, s), 5.86 (1H, s),
5.91 (1H, dd, J=16.2, 7.6 Hz), 6.83 (2H, d, J=8.7 Hz), 7.16 (2H, d,
J=8.7 Hz), 7.30-7.35 (6H, m), 9.43 (1H, d, J=7.6 Hz); .sup.13C NMR
(75 MHz, CDCl.sub.3) .delta. 14.1, 21.7, 22.6, 24.0, 24.6, 28.9,
29.0, 31.7, 32.6, 34.5, 36.2, 47.5, 51.3, 51.5, 55.4, 69.2, 71.3,
71.8, 74.4, 76.3, 102.7, 114.1, 118.2, 127.1, 127.7, 127.9, 128.7,
129.5, 130.7, 138.9, 151.6, 159.6, 166.6, 167.3, 172.1, 195.0.
[0376] To a solution of the dimethylacetal myristate (11 mg, 0.01
mmol) in 0.6 mL 1% aqueous CH.sub.2Cl.sub.2 was added solid
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 4 mg, 0.02 mmol) at
rt. The mixture was stirred for 2 h, pipetted directly onto a
column of silica gel, and the product eluted with 35% EtOAc/hexanes
to provide the corresponding intermediate alcohol (8 mg, 89%) as a
colorless oil: R.sub.f (35% EtOAc/hexanes)=0.22; IR 3528, 2930,
2858, 1745, 1720, 1686, 1458, 1437, 1380 cm.sup.-1; .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 0.86 (3H, t, J=6.9 Hz), 1.13 (3H, s),
1.17 (3H), 1.25 (10H, m), 1.52 (2H, m), 1.71 (2H, m), 2.12 (2H, m),
2.35 (1H, t, J=14.1 Hz), 2.60 (1H, d, J=3.6 Hz), 3.41 (3H, s), 3.45
(1H, m), 3.68 (3H, s), 3.82 (1H, s), 4.24 (1H, m), 4.59 (2H, ABq,
J=11.4 Hz), 5.47 (1H, s, C20), 5.86 (1H, s), 5.91 (1H, dd, J=15.9,
7.5 Hz), 7.29 (1H, d, J=15.9 Hz), 7.34 (5H, m), 9.52 (1H, d, J=7.5
Hz); .sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. 13.86, 15.44, 21.70,
22.38, 23.61, 24.39, 28.75, 28.81, 31.45, 32.75, 34.22, 39.50,
47.35, 51.13, 51.23, 68.32, 70.89, 71.06, 71.12, 78.51, 102.36,
117.43, 126.83, 127.97, 127.98, 128.57, 138.11, 151.80, 166.42,
167.11, 171.76, 194.73; [.alpha.].sub.D.sup.20=-21.0.degree. (c
1.0, CH.sub.2Cl.sub.2).
[0377] The intermediate alcohol (7 mg, 0.02 mmol) was dissolved in
1.1 mL CH.sub.3CN/H.sub.2O (9:1) and treated with 48% aqueous HF
(200 .mu.l, 300 mol % excess) at rt. The resulting mixture was
stirred for 1 h, quenched with sat. NaHCO.sub.3 and diluted with 10
mL EtOAc. The aqueous layer was separated and extracted with EtOAc
(2.times.). The combined organics were dried over Na.sub.2SO.sub.4
and concentrated in vacuo to afford the crude hemi-ketal enal (44
in Reaction Scheme 11, which provides the compound number
references for the remainder of the present example) as a colorless
oil. The crude product was purified by column chromatography on
silica gel with 35% EtOAc-hexanes as eluant affording 6 mg (86%) of
enal 44. R.sub.f (35% EtOAc/hexanes)=0.15; IR 3528, 2930, 2858,
1745, 1720, 1686, 1458, 1437, 1380 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 0.87 (3H, t, J=6.9 Hz), 1.13 (3H, s), 1.15 (3H,
s), 1.28 (10H, m), 1.52 (2H, m), 1.71 (2H, m), 2.12 (2H, m), 2.35
(1H, t, J=14.1 Hz), 2.60 (1H, d, J=3.6 Hz), 3.41 (3H, s), 3.45 (1H,
m), 3.68 (3H, s), 3.82 (1H, s), 4.24 (1H, m), 4.56 (2H, ABq, J=11.0
Hz), 5.47 (1H, s), 5.86 (1H, s), 5.91 (1H, dd, J=15.9, 7.4 Hz),
7.28 (1H, d, J=15.9 Hz), 7.35 (5H, s), 9.52 (1H, d, J=7.4 Hz);
.sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. 13.9, 14.1, 19.4, 20.0,
22.7, 23.0, 24.4, 24.6, 29.1, 29.1, 29.4, 29.4, 29.6, 29.7, 31.2,
31.4, 31.9, 32.4, 34.6, 36.0, 40.0, 42.6, 42.9, 45.0, 51.1, 64.5,
66.3, 68.7, 70.4, 73.7, 74.1, 75.8, 76.1, 77.2, 78.7, 94.0, 98.9,
102.4, 119.7, 125.7, 142.8, 151.8, 167.0, 172.1, 172.5, 194.7.
[0378] Carboxylic acid 6 (6 mg, 0.01 mmol) and Et.sub.3N (6 .mu.L,
0.04 mmol) were dissolved in 300 .mu.L toluene and treated with
2,4,6-trichlorobenzoylchloride (2.0 .mu.L, 0.01 mmol) dropwise at
rt. After 1 h at rt, a toluene solution of freshly prepared enal 44
and 4-dimethylaminopyridine (5 mg, 0.04 mmol) was added gradually
and stirring was continued for 40 min. The crude mixture was
pipetted directly onto a column of silica gel and the product
eluted with 20% EtOAc/hexanes to provide ester-enal 46 as a
colorless oil (9 mg, 90%). R.sub.f (35% EtOAc/hexanes)=0.71; IR
3487, 2927, 2856, 1723, 1689, 1455, 1379, 1228, 1156, 1113, 1084,
1032, 981 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.80
(1H, t, J=12.7 Hz), 0.93 (3H, t, J=7.0 Hz), 0.98 (3H, d, J=6.6 Hz),
1.10-1.41 (24H, m), 1.42-1.85 (21H, m), 1.92-2.13 (6H, m), 2.39
(1H, t, J=12.7 Hz), 2.81 (1H, d, J=13.2 Hz), 2.93 (1H, m), 3.05
(1H, m), 3.29 (3H, s), 3.33 (1H, s), 3.37-3.48 (2H, m), 3.80 (1H,
dd, J=11.4, 5.1 Hz), 3.95-4.05 (2H, m), 4.15 (1H, td, J=10.8, 0.9
Hz), 4.23 (1H, d, J=13.5 Hz), 4.46 (2H, ABq, J=11.0 Hz), 5.57 (1H,
s), 5.64 (1H, dd, J=10.6, 4.6 Hz), 6.05 (1H, dd, J=15.9, 7.5 Hz),
6.39 (1H, s), 7.10-7.35 (5H, m), 7.45 (1H, d, J=15.9 Hz), 9.60 (1H,
d, J=7.5 Hz); .sup.13C-NMR (75 MHz, CDCl.sub.3) .delta. -9.6, -9.4,
9.2, 10.3, 13.2, 14.1, 15.1, 19.3, 20.1, 21.3, 22.3, 22.4, 22.7,
23.8, 24.0, 24.6, 24.6, 29.0, 29.0, 29.3, 31.3, 31.6, 31.8, 34.2,
34.5, 35.2, 35.7, 36.2, 37.6, 43.5, 45.7, 51.6, 59.1, 64.9, 66.7,
71.1, 72.0, 72.9, 74.1, 75.3, 77.1, 100.2, 100.6, 121.2, 127.2,
127.3, 127.9, 128.6, 138.9, 151.2, 164.5, 166.3, 171.5, 175.1,
193.2; [.alpha.].sup.20.sub.D -19.degree. (c 1.5,
CH.sub.2Cl.sub.2).
[0379] To ester-enal 46 (8.0 mg, 0.001 mmol) in THF (0.5 mL) was
added 70% HF/pyridine (0.3 mL, 0.3 mmol) and stirred for 2 hours.
The reaction was then quenched with a saturated solution of sodium
bicarbonate. The biphasic mixture was extracted with ethyl acetate
(.times.4) and the combined organics were dried over sodium
sulfate. The solvent was removed in vacuo to provide crude
macrocycle. The crude mixture was chromatographed on silica gel and
the product was eluted with 50% EtOAc/hexanes to afford 5.0 mg
(83%) of the corresponding macrocycle as an clear oil: R.sub.f (40%
EtOAc/hexanes)=0.19; IR 3522, 2927, 2857, 1724, 1664, 1230, 1158,
1136, 1107, 979 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 0.84 (3H, t, J=5.4 Hz), 0.88-0.96 (5H, m), 1.00 (3H, d,
J=4.8 Hz), 1.02-1.55 (27H, m), 1.63-1.81 (2H, m), 1.82-1.94 (2H,
m), 2.03 (1H, br t, J=5.2 Hz), 2.19-2.27 (1H, m), 2.34 (1H, dt,
J=9, 1.5 Hz), 2.94-3.01 (2H, m), 3.22 (1H, s), 3.58 (1H, br d,
J=3.6 Hz), 3.68-3.74 (1H, m), 3.84-3.88 (1H, m), 3.94 (1H, dd,
J=8.6, 3.1 Hz), 4.23 (1H, dd, J=10.4, 1.7 Hz), 4.31 (1H, br t,
J=8.1 Hz), 4.56 (2H, ABq, J=11.0 Hz), 5.36-5.41 (1H, m), 5.50 (1H,
s), 5.61 (1H, d, J=5.4 Hz), 6.00 (1H, dd, J=12.0, 5.4 Hz), 6.36
(1H, s), 6.53 (1H, d, J=12.0 Hz); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. .delta.14.1, 19.4, 20.0, 22.7, 23.0, 24.4,
24.6, 29.1, 29.1, 29.4, 29.4, 29.6, 29.7, 31.2, 31.4, 31.9, 32.4,
34.6, 36.0, 40.0, 42.6, 42.9, 45.0, 51.1, 64.5, 66.3, 68.7, 70.4,
73.7, 74.1, 75.8, 76.1, 77.2, 78.7, 94.0, 98.9, 102.4, 119.7,
125.7, 142.8, 151.8, 167.0, 172.1, 172.5.
[0380] To 5.0 mg (0.0005 mmol) of crude macrocycle of the preceding
step in ethyl acetate (1.0 ml) was added a catalytic amount of
Pearlman's catalyst. The flask was evacuated and refilled with a 1
atm. hydrogen atmosphere (.times.4), stirred under hydrogen for 30
min, and then pipetted directly onto a silica gel column and eluted
with 60% ethyl acetate/hexanes. This process afforded 4.8 mg (99%)
of analogue 48 (702.4) (Formula II where R.sup.3 is OH, R.sup.20 is
--O--CO--C.sub.13H.sub.27, R.sup.21 is .dbd.CH--CO.sub.2Me,
R.sup.26 is methyl and X is oxygen) as an amorphous solid.
R.sub.f(50% EtOAc/hexanes)=0.16; IR 3522, 2927, 2857, 1724, 1664,
1230 IR (neat)=3455, 3319, 2929, 2856, 1735, 1380, 1230, 1138, 976
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.37 (3H, bs),
0.79-0.92 (14H, m), 1.06 (3H, d, J=6.4 Hz), 1.07 (1H, t, J=11.0
Hz), 1.10-1.25 (5H, m), 1.27 (3H, s), 1.50 (3H, s), 1.57-1.78 (4H,
m), 2.03 (2H, t, J=7.4 Hz), 2.17 (1H, dd, J=9.9/0.5 Hz), 2.37 (1H,
m), 2.40 (1H, m), 2.85 (1H, t, J=11.2 Hz), 2.96 (1H, t, J=10.8 Hz),
3.15 (3H, s), 3.68-3.72 (3H, m), 3.91 (1H, dd, J=11.2, 4.0 Hz),
4.13 (1H, t, J=9.7 Hz), 4.35 (1H, dd, J=13.9/2.2 Hz), 4.48 (1H, td,
J=11.0, 2.8 Hz), 4.70 (1H, d, J=12.1 Hz), 5.44 (1H, quint., J=4.8
Hz), 5.54 (1H, d, J=7.3 Hz), 5.69 (1H, s), 5.76 (1H, s), 5.85 (1H,
dd, J=16.1, 7.5 Hz), 6.40 (1H, d, J=1.8 Hz), 6.50 (1H, d, J=15.9
Hz); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 14.1, 19.4, 20.0,
22.7, 23.0, 24.4, 24.6, 29.1, 29.1, 29.4, 29.4, 29.6, 29.7, 31.2,
31.4, 31.9, 32.4, 34.6, 36.0, 40.0, 42.6, 42.9, 45.0, 51.1, 64.5,
66.3, 68.7, 70.4, 73.7, 74.1, 75.8, 76.1, 77.2, 78.7, 94.0, 98.9,
102.4, 119.7, 125.7, 142.8, 151.8, 167.0, 172.1, 172.5.
4D. Benzoate C20 Ester (702.5)
[0381] Enal 45 was prepared following the procedure for compound
III in Example 1C except that benzoic acid was substituted for
octanoic acid, to form the corresponding protected benzoate
product.
[0382] Carboxylic acid 6 (6 mg, 0.01 mmol) and Et.sub.3N (6 .mu.L,
0.04 mmol) were dissolved in 300 .mu.L toluene and treated with
2,4,6-trichlorobenzoylchloride (2 .mu.L, 0.01 mmol) dropwise at rt.
After 1 h at rt, a toluene solution of freshly prepared 45 and
4-dimethylaminopyridine (5 mg, 0.01 mmol) was added gradually and
stirring was continued for 40 min. The crude mixture was pipetted
directly onto a column of silica gel and the product eluted with
20% EtOAc/hexanes to provide the expected ester product as a
colorless oil (8 mg, 89%). R.sub.f (35% EtOAc/hexanes)=0.71; IR
3460, 2927, 2856, 1723 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 0.083H, s), 0.08 (3H, s), 0.81 (12H, m), 0.82-0.96 (3H, m),
1.06-1.32 (15H, m), 1.59-1.81 (4H, m), 2.20 (1H, t, J=9.3 Hz),
3.33-3.44 (2H, m), 3.67-3.84 (1H, m), 4.00-4.15 (4H, m), 4.31-4.39
(1H, m), 4.55 (2H, ABq, J=8.5 Hz), 5.41 (1H, s), 5.75 (1H, dd,
J=15.5, 3.4 Hz), 6.01 (1H, s), 6.39 (1H, s), 7.28-7.47 (9H, m),
7.84 (1H, d, J=6.9 Hz), 9.17 (1H, d, J=7.3 Hz); .sup.13C-NMR (75
MHz, CDCl.sub.3) .delta. -9.6, -9.4, 9.2, 10.3, 13.2, 14.2, 18.5,
22.0, 23.5, 28.4, 28.7, 30.7, 31.5, 33.5, 39.1, 41.5, 41.9, 44.0,
50.1, 63.7, 65.3, 67.8, 69.6, 70.3, 73.8, 74.8, 75.1, 76.2, 77.7,
98.1, 101.3, 118.8, 124.7, 126.7, 127.4, 127.5, 128.9, 132.2,
137.3, 141.8, 150.8, 163.6, 165.9, 170.2.
[0383] To the ester of the preceding step (13 mg, 0.02 mmol) in THF
(0.5 mL) was added pyridine (360 .mu.L, 0.45 mmol) followed by 70%
HF/pyridine (144 .mu.L, 500 mol % excess) with stirring for 20
hours. The reaction was then quenched with a saturated solution of
sodium bicarbonate. The biphasic mixture was extracted with ethyl
acetate (.times.4) and the combined organics were dried over sodium
sulfate. The solvent was removed in vacuo to provide the
corresponding crude C3 hydroxyester. The crude mixture was pipetted
directly onto a column of silica gel and the product eluted with
35% EtOAc/hexanes to provide the purified C3 hydroxyester as a
colorless oil (9 mg, 82%). R.sub.f (40% EtOAc/hexanes)=0.19; IR
3522, 2927, 2857, 1724 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3)
0.81-0.91 (3H, m), 1.08 (3H, s), 1.20-1.59 (9H, m), 1.31 (6H, s),
1.92-2.18 (4H, m), 2.45 (2H, bs), 3.40-3.58 (2H, m), 3.67 (3H, s),
3.69-3.78 (2H, m), 3.88-3.98 (2H, m) 4.03-4.24 (3H, m), 4.45 (1H,
d, J=9.2 Hz), 4.65 (2H, ABq, J=8.5 Hz), 5.17 (1H, d, J=9.7 Hz),
5.22 (1H, s), 5.40 (1H, s), 5.44 (1H, dd, J=15.5, 7.3 Hz), 6.06
(1H, s), 6.08 (1H, d, J=15.5 Hz), 7.27-7.59 (9H, m), 8.05 (1H, d,
J=6.9 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 14.2, 18.5,
22.0, 23.5, 28.4, 28.7, 30.7, 31.5, 33.5, 39.1, 41.5, 41.9, 44.0,
50.1, 63.7, 65.3, 67.8, 69.6, 70.3, 73.8, 74.8, 75.1, 76.2, 77.7,
98.1, 101.3, 118.8, 124.7, 126.7, 127.4, 127.5, 128.9, 132.2,
137.3, 141.8, 150.8, 163.6, 165.9, 170.2;
[.alpha.].sup.20.sub.D-11.5 (c 0.9, CDCl.sub.3).
[0384] To 4.0 mg (0.001 mmol) of the crude C3 hydroxyester of the
preceding step in ethyl acetate (1.0 ml) was added a catalytic
amount of Pearlman's catalyst. The flask was evacuated and refilled
with a 1 atm. hydrogen atmosphere (.times.4). Stirred under
hydrogen for 30 min. and then pipetted directly onto a silica gel
column and eluted with 60% ethyl acetate/hexanes. HPLC (hexane:
methylene chloride: i-propanol, 16:3:1) Isolated 2.2 mg (63%) of
analogue 49 (702.5) (Formula II where R.sup.3 is OH, R.sup.20 is
--O--CO-Ph, R.sup.21 is .dbd.CH--CO.sub.2Me, R.sup.26 is methyl and
X is oxygen) as an amorphous solid. R.sub.f (50%
EtOAc/hexanes)=0.16; IR 3522, 2927, 2857, 1724, 1664, 1230 IR
(neat)=3455, 3319, 2929, 2856, 1735, 1380, 1230, 1138, 976
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.81-0.92 (3H,
m), 1.08 (3H, s), 1.20-1.59 (9H, m), 1.32 (6H, s), 1.90-2.18 (4H,
m), 2.52-2.56 (2H, m), 3.40-3.55 (2H, m), 3.67 (3H, s), 3.73-3.79
(2H, m), 3.82-3.95 (2H, m) 4.02-4.22 (2H, m), 4.51 (1H, d, J=9.1
Hz), 5.11 (1H, d, J=8.9 Hz), 5.26 (1H, s), 5.40 (1H, s), 5.42 (1H,
dd, J=15.5, 3.4 Hz), 6.04 (1H, d, J=15.5 Hz), 6.07 (1H, s),
7.34-7.57 (4H, m), 8.04 (1H, d, J=7.3 Hz); .sup.13C NMR (125 MHz,
CDCl.sub.3) .delta. 13.1, 18.4, 18.9, 21.7, 23.5, 28.7, 30.6, 31.4,
35.0, 39.1, 41.6, 50.1, 63.6, 67.7, 69.2, 72.6, 76.1, 77.7, 93.0,
99.2, 113.0, 124.7, 127.2, 127.5, 128.9, 132.2, 146.3, 148.1,
149.9, 150.6, 171.4, 173.2; [.alpha.].sup.25.sub.D=-7.0.degree.
(c=0.36, CDCl.sub.3).
Example 5
Protein Kinase C (Isozyme Mix) Assay Protocol
[0385] The following procedure was used, based on a modification of
a previous procedure described by Tanaka et al. (1986). Filters
(Whatman GF-B, 21 mm diam.) are soaked for 1 h in a solution
containing deionized water (97 mL), and 10% polyethyleneamine (3
mL). A filtering buffer solution containing TRIS (1M, pH 7.4, 10
mL) and water (490 mL) is prepared and cooled on ice. An assay
buffer solution is prepared by the addition of TRIS (1M, pH 7.4, 1
mL), KCl (1M, 2 mL), CaCl.sub.2 (0.1M, 30 .mu.L), bovine serum
albumin (40 mg), diluted to 20 .mu.L with deionized water and
stored on ice. Phosphatidyl serine vesicles are prepared by the
addition of phosphatidyl serine (10 mg/mL in chloroform, 0.4 mL) to
a glass test tube followed by removal of the chloroform under a
stream of nitrogen (5 min). To this viscous liquid is added a
portion of the prepared assay buffer (4 mL) and the resulting
mixture is then transferred to a plastic tube with washing. This
tube is then sonicated (Branson Sonifier 250, power=6, 40% duty
cycle) four times for 30 sec. with a 30 sec. rest period between
sonications. The resulting solution is stored over ice. PKC is
prepared by addition of cooled assay buffer (10 mL) to PKC (25
.mu.L) purified from rat brain by the method of Mochly-Rosen and
Koshland (1986) and then stored on ice. Stock solutions of
compounds are diluted with absolute ethanol in glass in serial
fashion. Each plastic assay incubation tube is made to contain
prepared phosphatidyl serine vesicles (60 .mu.L), prepared PKC
solution (200 .mu.L) and analogue (0-20 .mu.L) plus EtOH (20-0
.mu.L) for a total volume of 20 .mu.L). Lastly, tritiated phorbol
12,13-dibutyrate (PDBU) (30 nM, 20 .mu.L) is added to each tube.
The assay is carried out using 7-10 analogue concentrations, each
in triplicate. Non-specific binding is measured in 1-3 tubes by the
substitution of phorbol myristate acetate (PMA) (1 mM, 5 .mu.L) and
EtOH (15 .mu.L) for the analogue/EtOH combination. The tubes are
incubated at 37.degree. C. for 90 min. and then put on ice for 5
min. Each tube is then filtered separately through a pre-soaked
filter disc. Each tube is rinsed with cold 20 mM TRIS buffer (500
.mu.L) and the rinseate is added to the filter. The filter is
subsequently rinsed with cold 20 mM TRIS buffer (5 mL) dropwise.
The filters are then put in separate scintillation vials and
Universol.COPYRGT. scintillation fluid is added (3 mL). The filters
are immediately counted in a scintillation counter (Beckman LS
6000SC). Counts per minute are averaged among three trials at each
concentration. The data is then plotted using a least squares fit
algorithm with the Macintosh version of Kaleidagraph.COPYRGT.
(Abelbeck Software) and an IC.sub.50 (defined as the concentration
of analogue required to displace half of the specific PDBU binding
to PKC) is calculated. The IC.sub.50 then allows determination of
the K; for the analogue from the equation:
K.sub.i=IC.sub.50/(I+[PDBu]/K.sub.d of PDBu). The K.sub.d of
[H.sup.3]-PDBu was determined under identical conditions to be 1.55
nM.
Example 6
PKC.delta.-C1B Assay Protocol
[0386] All aspects of the PKC.delta.-C1B assay are identical to the
PKC isozyme mix assay from Example 5 except the following features:
In the PKC.delta.-C1B assay system, assay buffer is made without
CaCl.sub.2. PKC.delta.-C1B (200 .mu.g, 34.14 nmol), prepared by the
method of Wender et al. (1995) and Irie et al. (1998) is dissolved
in deionized water (160 .mu.L) and ZnCl.sub.2 (5 mM, 40 .mu.L) is
added. The resulting solution is allowed to stand at 4.degree. C.
for 10 min. An aliquot (10 .mu.L) of this solution is diluted to 2
mL with deionized water. An aliquot (290 .mu.L) is further diluted
to 20 mL with assay buffer and is ready for use. The incubation
time is shortened from 90 min. to 30 min. Lastly, during the
filtering portion of the assay, the tube is not washed with
filtering buffer (0.5 mL).
[0387] When tested as described above, the C26 desmethyl analogue
702.1 (Example 3A), had significantly higher activity than the
corresponding C26 methyl-containing analogue (Formula 1998a where
R.sup.3 is OH). Similarly, among several analogues having different
C20 ester groups, the presence of longer R.sup.20 substituents (48)
or an aryl substituent (49) also afforded higher activity. The
results are shown below in Table I (in all compounds tested,
R.sup.3 was OH and R.sup.21 was .dbd.CH--CO.sub.2Me).
TABLE-US-00001 TABLE 1 PKC.delta.-C1B Assay Compound R.sup.20
R.sup.26 Ki (nM) Phorbol dibutyrate 1.7 (K.sub.d value) Formula
1998a --OC(O)C.sub.7H.sub.15 CH.sub.3 5.1 Formula IIa (702.1)
--OC(O)C.sub.7H.sub.15 H 0.30 .+-. 0.07 Formula 702.2
--OC(O)CH.sub.3 CH.sub.3 232 .+-. 11 Formula 702.3
--OC(O)C.sub.6H.sub.13 CH.sub.3 35 Formula 702.4
--OC(O)C.sub.13H.sub.27 CH.sub.3 1.3 Formula 702.5 --OC(O)Phenyl
CH.sub.3 1.7
Example 7
P388 Murine Lymphocytic Leukemia Cell Assay
[0388] Cells from a P388 cell line (CellGate, Inc., Sunnyvale,
Calif.) are grown in RPMI 1640 cell medium containing fetal calf
serum (10%), L-glutamine, penicillin, streptomycin and are split
twice weekly. All compounds are first diluted with DMSO. Later
serial dilutions are done with a phosphate buffer solution (HYQ
DPBS modified phosphate buffered saline). All dilutions are done in
glass vials and the final DMSO concentration is always below 0.5%
by volume. Final two-fold dilutions are done in a 96 well plate
using cell media so that each well contains 50 .mu.L. All compounds
are assayed in quadruplicate over 12 concentrations. Cell
concentration is measured using a hemacytometer and the final cell
concentration is adjusted to 1.times.10.sup.4 cells/mL with cell
medium. The resulting solution of cells (50 .mu.L) is then added to
each well and the plates are incubated for 5 days in a 37.degree.
C., 5% CO.sub.2, humidified incubator (Sanyo CO.sub.2 incubator).
MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide, 10 .mu.L) is then added to each well and the plates are
re-incubated under identical conditions for 2 h. To each well is
then added acidified isopropanol (150 .mu.L of i-PrOH solution
containing 0.05 N HCl) and mixed thoroughly. The plates are then
scanned at 595 nm and the absorbances are read (Wallac Victor 1420
Multilabel Counter). The resulting data is then analyzed to
determine an ED.sub.50 value using the Prism software package
(GraphPad).
[0389] When tested as described above, the C26 desmethyl analogue
702.1 (Example 3A), had significantly higher activity than the
corresponding C26 methyl-containing analogue. Similarly, among
several analogues having different C20 ester groups, the presence
of longer R20 substituents (48) or an aryl substituent (49) also
afforded higher activity. The results are shown below in Table 2
(in all compounds tested, R.sup.3 was OH and R.sup.21 was
.dbd.CH--CO.sub.2Me). TABLE-US-00002 TABLE 2 P388 Assay Compound
R.sup.20 R.sup.26 ED.sub.50 (nM) Formula 1998a
--OC(O)C.sub.7H.sub.15 CH.sub.3 76 Formula IIa (702.1)
--OC(O)C.sub.7H.sub.15 H 17 Formula 702.2 --OC(O)CH.sub.3 CH.sub.3
181 Formula 702.3 --OC(O)C.sub.6H.sub.13 CH.sub.3 38 Formula 702.4
--OC(O)C.sub.13H.sub.27 CH.sub.3 3.6 Formula 702.5 --OC(O)Phenyl
CH.sub.3 42
Example 8
In Vitro Inhibition of Growth in Human Cancer Cell Lines
[0390] Anticancer data were obtained in vitro for C26 desmethyl
bryostatin analogue 702.1 (Example 3A) tested against a spectrum of
different NCI human cancer cell-lines associated with various
cancer conditions. The results are shown in Table 3. Data obtained
with Bryostatin-1 are included for comparison. Growth inhibition
(GI50) values are expressed as the log of molar concentration at
half-maximum inhibition. As can be seen, the C26 desmethyl compound
was at least as potent, on average, as bryostatin-1 for all cell
groups tested. Moreover, the C26 desmethyl compound was more active
than bryostatin-1 by more than 2 orders of magnitude for several
cell lines: K-562 and MOLT-4 (leukemia), NCI-H460 (NSC lung),
HCC-2998 (colon), TK-10 (renal), and MDA-MB-435 (breast). These
results are significant and surprising since the C27 methyl group
attached to C26 was previously believed to be necessary for
activity. TABLE-US-00003 TABLE 3 Cell Line: desmethyl Bryo-1
Difference Leukemia CCRF-CEM -5.81 -5.30 -0.51 HL-60(TB) -5.84
-5.70 -0.14 K-562 -7.5 -5.40 -2.10 MOLT-4 <-8.0 -5.50 -2.50
RPMI-8226 -5.82 >-5 -0.82 SR -5.1 >-5 -0.10 NSC Lung
A549/ATCC -6.62 -5.20 -1.42 EKVX -5.58 -5.30 -0.28 HOP-62 -4.79
>-5 HOP-92 -4.67 -5.30 0.63 NCI-H226 >-5 NCI-H23 >-5
NCI-H322M -4.38 -6.00 1.62 NCI-H460 <-8 -5.60 -2.40 NCI-H522
>-5 Colon COLO 205 -7.08 -5.40 -1.68 HCC-2998 -7.54 -5.30 -2.24
HCT-116 -5.32 -5.30 -0.02 HCT-15 -4.76 >-5 HT29 -5.55 -5.30
-0.25 KM12 -5.34 -5.20 -0.14 SW-620 -5.12 -5.50 0.38 CNS SF-268
-4.97 -5.10 0.13 SF-295 -6.05 -5.20 -0.85 SF-539 -5.77 >-5 -0.77
SNB-19 -5.2 >-5 -0.20 SNB-75 -4.97 -5.50 0.53 U251 -5.4 -5.10
-0.30 Prostate PC-3 -5.6 -5.30 -0.30 DU-145 -5.02 >-5 -0.02
Melanoma LOX IMVI -5.47 -5.10 -0.37 MALME-3M -5.20 MI4 -5.26 >-5
-0.26 SK-MEL-2 -5.02 -5.20 0.18 SK-MEL-28 -4.67 -5.10 0.43 SK-MEL-5
-6.43 -5.70 -0.73 UACC-257 -5.13 -5.10 -0.03 UACC-62 -5.11 -5.30
0.19 Ovarian IGROVI -4.88 -5.30 0.42 OVCAR-3 -4.83 -5.10 0.27
OVCAR-4 -5.15 -5.50 0.35 OVCAR-5 -5.28 >-5 -0.28 OVCAR-8 -4.77
-5.10 0.33 SK-OV-3 -4.3 -5.10 0.80 Renal 786-0 -5.46 -5.20 -0.26
A498 -6.38 >-5 -1.38 ACHN -5.94 -5.50 -0.44 CAKI-1 -5.7 -5.40
-0.30 RXF-393 -5.30 SN12C -5.59 -5.10 -0.49 TK-10 -7.03 >-5
-2.03 UO-31 -4.85 -5.60 0.75 Breast MCF7 -5.4 -5.20 -0.20
NCI/ADR-RES -4.74 >-5 MDA-MB- -5.69 -5.20 -0.49 231/ATCC
MDA-MB-435 -7.66 -5.10 -2.56 MDA-N -5.10 BT-549 -4.71 -5.10 0.39
T-47D -5.02 -5.20 0.18 HS 578T -5.18 -5.2 0.02
Example 9
Formula 405 where X is --O--
[0391] ##STR86##
[0392] In an oven dried argon purged 100 mL round-bottom flask
charged with a magnetic stir bar was added a solution of 482 mg
(1.49 mmol, 1.0 eq) of the compound of Formula 404.1 in 31 mL of
isobutylvinyl ether. 237 mg (0.744 mmol, 0.5 eq) of Hg(II)
diacetate was added in one portion at room temperature [rt]. The
reaction was run at rt for 48 hrs. The reaction was diluted with
EtOAc and washed with sat. NaHCO.sub.3 and brine. The combined
organic layers were dried over MgSO.sub.4, filtered and
concentrated. Rapid flash chromatography using 15% ethyl acetate,
85% petroleum ether plus 1% triethyl amine yielded crude vinylated
pyran. This was carried on immediately.
[0393] In an oven-dried argon-purged 250 mL round-bottom flask
charged with a magnetic stir bar was added a solution of 480 mg
(1.37 mmol, 1.0 eq) of the vinylated pyran in 26 mL of degassed,
(via bubbling argon gas through), 99%+anhydrous decane (Aldrich).
The reaction vessel was lowered into a preheated 155.degree. C. oil
bath. The reaction was heated for 3 hrs at this temperature under
an argon atmosphere. It was then removed from the oil bath and
cooled to rt. It was allowed to sit overnight under an argon
atmosphere. The whole reaction was loaded onto a column using
petroleum ether to assist in the transfer. The column was then
eluted with 10% ethyl acetate 90% petroleum ether, to yield 431 mg
(83% over two steps) of the title compound of Formula 405,
[6-(7-isopropyl-10-methyl-1,5-dioxa-spiro[5,5]undec-2-ylmethyl)-5,6-dihyd-
ro-2H-pyran-2-yl]-acetaldehyde, as a clear colorless oil.
Example 10
[0394] ##STR87## Procedure:
[0395] 6.38 g of freshly distilled diisopropyl amine was combined
with 100 ml of dry THF and cooled to -78.degree. C. 40.7 mL of 1.55
M n-BuLi in Hexanes was then added slowly. After 10 minutes at
-78.degree. C., the solution was warmed to 0.degree. C. for 15
minutes then re-cooled to -78.degree. C. To this solution of
lithium diisopropylamide [LDA] at -78.degree. C. was added 5 g of
tert-butylacetoacetate slowly. After 10 minutes at -78.degree. C.,
the solution was warmed to 0.degree. C. for 40 minutes. The bright
yellow solution of dianion was then cooled to -78.degree. C. and
2.97 g of diethylglutarate was added in 5 mL of dry THF in one
portion. After 30 minutes at -78.degree. C., the reaction was
quenched by the addition of 140 mL of 1 N HCl in water. The
reaction was then partitioned between the aqueous layer and 200 mL
of ether. The ether was removed in vacuo and the residue purified
via flash chromatography (25% Ether:Pentane-Rf=0.3) to give 65%
product. ##STR88## Procedure:
[0396] A 2-neck flask with 2 gas flow adaptors was charged with 21
mg of COD-RuBis-2-methylallyl complex and 50 mg of (S)--BINAP. To
this was added 4 mL of degassed acetone (Argon purge for 20 min.)
and 12 mg of HBr (24 .mu.L of 49% soln) in 0.4 mL degassed MeOH (as
above). The solution became red-orange with a red precipitate.
After 30 minutes, the solvents were carefully remove in vacuo (air
sensitive!) to yield a tan powder that was used directly. To the
crude (S)--BINAP--RuBr.sub.2 was added 1 g of 3,5-dioxo-nonanedioic
acid 1-tert-butyl ester 9-ethyl ester in 10 mL EtOH. The solution
was stirred rapidly and the suspension transferred to a Parr Bomb
apparatus under Argon blanket. The bomb was sealed and pressurized
to 20 atmospheres for 3 cycles then left at 20 atmospheres and
heated to 75.degree. C. with stirring. After 6 hours, the reaction
appeared complete by TLC and the product isolated by Flash
Chromatography (Rf=0.2 in 1:1 Ethyl Acetate:Pentane) to yield 442
mg of product that was recrystallized in Ethyl Acetate:Pentane to
yield 320 mg product. ##STR89## Procedure:
[0397] 126 mg of 3,5-dihydroxy-nonanedioic acid 1-tert-butyl ester
9-ethyl ester was dissolved in 16 mL of dry toluene then cooled to
-10.degree. C. in an acetone/ice bath. To this was added 4 mg of
Tosic Acid. After 8 hours at -10.degree. C., the S.M. appeared
consumed. The reaction was quenched with saturated sodium
bicarbonate solution and partitioned between ethyl acetate and
saturated sodium bicarbonate solution. The ethyl acetate was
removed in vacuo and the residue purified via flash chromatography
(1:1 Ethyl Acetate:Pentane->1.5:1 Ethyl Acetate:Pentane Rf=0.3
in 1.5:1 E.A.:Pentane). Yield=75% ##STR90## Procedure:
[0398] 29 mg of 3-hydroxy-4-(6-oxo-tetrahydro-pyran-2-yl)-butyric
acid tert-butyl ester was dissolved in 3 mL of dichloromethane and
cooled to 0.degree. C. To this was added 24 mg of dry 2,6-lutidine.
60 mg of TBS triflate was added followed by 50 mg of DMAP. The
solution was allowed to warm to r.t. and stirred overnight. The
reaction was then partitioned between ethyl acetate and saturated
bicarbonate solution. The ethyl acetate was removed in vacuo and
the residue purified via flash chromatography (15% ethyl
acetate:pentane Rf=0.3) to yield the TBS ether in 72% yield.
##STR91## Procedure:
[0399] To 190 mg of NaH (95%) in 80 mL of dry THF was added 1.05 g
of ethyl acetoacetate in 15 mL of dry THF at 0.degree. C. After 10
minutes, 5.4 mL of 1.5 M n-BuLi in Hexanes was added. After 10
additional minutes, 1% of the solution (.about.1 mL) was taken and
cooled to -78.degree. C. in a dry flask. To this was added 15 mg of
3-(tert-butyl-dimethyl-silanyloxy)-4-(6-oxo-tetrahydro-pyran-2-yl)-butyri-
c acid tert-butyl ester in 1 mL of dry THF. After 30 minutes, the
reaction was quenched with 1N HCl and partitioned into ethyl
acetate. The ethyl acetate was removed in vacuo and the residue
purified by flash chromatography (30% ethyl acetate:pentane
Rf=streak.about.0.3). ##STR92## Procedure:
[0400] 5 mg of
3-(tert-butyl-dimethyl-silanyloxy)-4-[6-(3-ethoxycarbonyl-2-oxo-propyl)-6-
-hydroxy-tetrahydro-pyran-2-yl]-butyric acid tert-butyl ester was
dissolved in 1 mL of dichloromethane and cooled to -78.degree. C.
To this was added 100 .mu.L of TES and 50 .mu.L of TFA. The
solution was allowed to slowly warm to 0.degree. C., at which time
a lower slightly lower R.sub.f spot cleanly formed (Rf=0.2 in 30%
E.A.:Pentane). The reaction was quenched with saturated bicarbonate
solution and extracted into ethyl acetate. The product was then
purified via flash chromatography to yield the syn tetrahydropyran
in 90% without the TBS ether.
Example 11
[0401] Compound and synthetic step references in this example
correspond to the following two schemes (as opposed to the reaction
schemes and formula numbers employed previously in the
specification). ##STR93## ##STR94##
[0402] To a stirred solution of diisopropylamine (16.82 ml, 120
mmol) in THF (50 ml) was added n-butyllithium (48 ml, 2.5 M in
hexane) dropwise at -78.degree. C. The mixture was stirred at
0.degree. C. for 30 min, cooled again to -78.degree. C., and
treated with methyl isobutyrate (22.5 ml, 109 mmol) slowly over 10
min. The reaction mixture was stirred for 1.5 hours at -78.degree.
C. and 1 hour at 40.degree. C. After addition of allyl bromide
(11.8 ml, 135 mmol) dissolved in THF (25 ml) the mixture was
allowed to warm up to room temperature [rt] overnight. The solution
was evaporated without aqueous workup. The formed solid LiBr was
removed by chromatographic filtration on silica gel with
ether/pentane (1:1). Fractional distillation gave 5 (12.77 g, 90%)
at bp=135-150.degree. C. as colorless liquid. ##STR95##
[0403] To a stirred solution of 5 (14.22 g, 101 mmol) in CCl.sub.4
(80 ml) was added N-bromosuccinimide (20 g, 112 mmol) and
dibenzoylperoxide (80 mg, 0.33 mmol) in a single portion. The
reaction mixture was heated at reflux for 2 hours using an
preheated oil bath (105.degree. C.). After cooling to rt, the
mixture was filtered and the residue was washed with CCl.sub.4. The
solvent was removed in vacuo and the crude material purified using
flash chromatography on silica gel with EtOAc/hexane (9/1) yielding
the desired allylic bromide 26 (8.34 g, 74%) as yellow oil.
##STR96##
[0404] To a suspension of sodium hydride (1.83 g, 45.8 mmol; 60% in
mineral oil) in 100 ml anhydrous THF was added a solution of
p-methoxy benzylalcohol (5.75 g, 41.6 mmol) in THF (25 ml) slowly
over 15 minutes at 0.degree. C. The mixture was stirred at rt for
45 minutes before a solution of the previously prepared allylic
bromide (2.3 g, 10.4 mmol) in 30 ml THF was added over 15 min. The
reaction mixture was warmed to 35.degree. C. for 6 h. The reaction
was cooled to rt and quenched with water carefully. The aqueous
layer was extracted with Et.sub.2O (2.times.), then neutralized
with 2N HCl, and again extracted with EtOAc (4.times.). The
combined organic layers were dried over MgSO.sub.4 and concentrated
in vacuo. Purification of the residue on silica gel (EtOAc/hexane
33%-80%) afforded 6b (2.03 g, 74%) as yellow oil.
[0405] Alternatively the reaction can be quenched with saturated
NH.sub.4Cl solution. The aqueous layer was then extracted with
Et.sub.2O (3.times.) and the combined layers were dried over
MgSO.sub.4 and concentrated in vacuo. The resulting oil was
purified by flash chromatography (EtOAc 20%) to provide a mixture
of 6a & 6b, the methyl and p-methoxybenzyl esters.
##STR97##
[0406] To a stirred solution of acid 6c (133 mg, 0.5 mmol) in
anhydrous Et.sub.2O (6 ml) and cooled to 0.degree. C., was added
sodium hydride (240 mg, 6.0 mmol; 60% in mineral oil) in a single
portion. The mixture was stirred for 30 minutes at 0.degree. C. and
then oxalyl chloride (0.26 ml, 3.0 mmol) was added in a single
portion. The resulting mixture was allowed to warm to rt and
stirring was continued for 2 h. The mixture was then concentrated
in vacuo and the resulting oil used without further purification.
##STR98##
[0407] To a stirred solution of methyl and p-methoxybenzyl esters
6a and 6b (6.56 g, 23.55 mmol) in THF (20 ml), was added
N,O-dimethylhydroxylamine hydrochloride (3.68 g, 37.7 mmol),
followed by dropwise addition of phenylmagnesium bromide (2M in
THF, 18.25 ml, 36.5 mmol) at -20.degree. C. To the reaction mixture
was subsequently added phenylmagnesium bromide (18.25 ml, 36.5
mmol) over 45 min. Stirring was continued for 1 hour at -10.degree.
C. The reaction was quenched with saturated NH.sub.4Cl and diluted
with Et.sub.2O. The aqueous layer was extracted with EtOAc
(3.times.) and the combined organics were dried over MgSO.sub.4 and
concentrated in vacuo. The resulting oil was purified by flash
chromatography (EtOAc/hexane 1/3) to provide Weinreb amide 8 (6.54
g, 90%) as colorless oil. ##STR99##
[0408] A solution of Weinreb amide 8 (5.82 g, 18.1 mmol) in THF
(100 ml) was cooled to -78.degree. C. then treated with
methyllithium (1.4 M in Et.sub.2O, 17.47 ml, 24.5 mmol). Stirring
was continued for 1 hour at -78.degree. C., and the reaction was
quenched with saturated NH.sub.4Cl, and diluted with Et.sub.2O. The
aqueous layer was extracted with EtOAc (3.times.) and the combined
organics were dried over MgSO.sub.4 and concentrated in vacuo. The
resulting oil was purified by flash chromatography (EtOAc/hexane
1/4) to provide methyl ketone 9 (4.89 g, 99%) as a colorless oil.
##STR100##
[0409] To a stirred solution of diisopropylamine (0.21 ml, 1.5
mmol) in THF (3 ml) was added n-butyllithium (0.93 ml, 1.6 M in
hexane) dropwise at -78.degree. C. The mixture was allowed to warm
to 0.degree. C. and stirred for 30 min, then cooled again to
-78.degree. C., and a solution of acetone (0.11 ml, 1.5 mmol) in
THF (1 ml) was added dropwise. The acetone used was dried over 4
.ANG. molecular sieves for several days and was dried again over 4
.ANG. molecular sieves in THF solution immediately prior to use.
After stirring for 20 minutes, the mixture was treated with a
solution of acid chloride 7 (0.5 mmol) in 2 ml THF and stirring was
continued at -78.degree. C. for 1 h. The reaction was quenched with
saturated NH.sub.4Cl and warmed up to rt and diluted with
Et.sub.2O. The aqueous layer was extracted with EtOAc (3.times.)
and the combined organic layers were dried over MgSO.sub.4 and
concentrated in vacuo. Purification of the residue by flash
chromatography on silica gel (EtOAc/hexane 1/2) yielded
.beta.-diketone 11 (117 mg, 77%) as an orange oil. ##STR101##
[0410] To a stirred solution of diisopropylamine (0.21 ml, 1.5
mmol) in THF (3 ml) was added n-butyllithium (0.93 ml, 1.6 M in
hexane) dropwise at -78.degree. C. The mixture was warmed to
0.degree. C. and stirred for 30 minutes, then cooled again to
-78.degree. C., and treated with a solution of acetone (0.11 ml,
1.5 mmol) in THF (1 ml). The acetone used was dried over 4 .ANG.
molecular sieves for several days and was dried again over 4 .ANG.
molecular sieves in THF solution immediately prior to use. After
recooling to -78.degree. C. and stirring for 20 minutes, aldehyde
10 (164 mg, 0.5 mmol) was added dropwise and stirring was continued
for 15 minutes. The reaction was quenched by addition of saturated
NH.sub.4Cl and the mixture was warmed to rt and diluted with
Et.sub.2O. The mixture was diluted with Et.sub.2O and the layers
separated. The aqueous layer was then extracted with EtOAc
(3.times.). The combined organics were washed with brine, dried
over MgSO.sub.4, and concentrated in vacuo. Flash chromatography on
silica gel (EtOAc/hexane 1/2) provided 12 (169 mg, 87%). The
diastereoselectivity was determined to be 81%, favoring the desired
isomer, after coupling with acid chloride 7 and cyclization to
pyranone 14. ##STR102##
[0411] From 11: To a stirred solution of diisopropylamine (1.19 ml,
8.48 mmol) in THF (13 ml) was added n-butyllithium (4.08 ml, 8.16
mmol, 2.0 M in hexane) dropwise at -78.degree. C. The mixture was
warmed to 0.degree. C. and stirred for 30 minutes, then a solution
of .beta.-diketone 11 (2.30 g, 8.77 mmol) in THF (13 ml) was added
slowly over 10 minutes. After stirring for 1 hour at 0.degree. C.,
the mixture was cooled to -78.degree. C. and aldeyde 2 (1.21 g,
3.69 mmol) was added in a single portion. Stirring was continued
for 30 minutes at -78.degree. C. and the reaction mixture was then
quenched with saturated NH.sub.4Cl solution, allowed to warm to rt,
and diluted with Et.sub.2O. The mixture was extracted with EtOAc
(3.times.) and the combined organics were dried over MgSO.sub.4 and
concentrated in vacuo. Flash chromatography on silica gel
(EtOAc/hexane 1/2) afforded the aldol 13 (1.51 g, 65%) as a orange
oil. The ratio of the two diastereomers was determined after
cyclization to pyranone 7, and was 1.45:1 favoring the desired
isomer.
[0412] From 12: To a stirred solution of diisopropylamine (0.46
.mu.L, 0.204 mmol) in THF (0.5 ml) was added n-butyllithium (0.128
ml, 0.204 mmol, 1.6M in hexanes) dropwise at -78.degree. C. The
mixture was warmed to 0.degree. C. and stirred for 30 min, then
cooled again to -78.degree. C., and treated with a solution of
.beta.-hydroxy ketone 12 (42.0 mg, 0.0662 mmol) in THF, (1 ml)
dropwise. After stirring for 20 minutes at -78.degree. C. the
mixture was treated with a solution of acid chloride 7 (0.5 ml,
.about.0.20 mmol, prepared from 0.5 mmol acid 6c dissolved in 1 ml
THF) and stirring was continued at -78.degree. C. for 30 min. The
reaction was quenched with H.sub.2O, warmed up to rt by stirring
vigorously for 30 min, and diluted with Et.sub.2O. The aqueous
layer was extracted with EtOAc (3.times.) and the combined organic
layers were dried over MgSO.sub.4 and concentrated in vacuo.
Purification of the residue by flash chromatography yielded 13
(41.9 mg, 61%).
[0413] From 28: To distilled pyridine (1.3 ml, 16.8 mmol) in a high
density polyethylene (HDPE) vial at -78.degree. C. was added
dropwise over 10 minutes a solution of 70% HFpyridine (0.5 ml,
.about.17.5 mmol HF, .about.4.4 mmol pyridine) to form a nearly
equimolar solution of HFpyridine. This solution was stored at
-20.degree. C. until needed.
[0414] To a solution of the previously prepared C23 OTBS
.beta.-diketone 28 (0.025 g, 0.033 mmol) in dry THF (2 ml) in a
HDPE vial was rapidly added the HFpyridine solution prepared above
(0.25 ml) in a single portion. The resulting solution was layered
with argon, sealed and stirred vigorously for 7 days at rt. The
vial was unsealed, and the reaction quenched with saturated aqueous
NaHCO.sub.3 (1 ml). The reaction was diluted with ethyl acetate,
the layers separated, and the aqueous phase extracted 3 times with
ethyl acetate. The combined organic fractions were pooled, dried
over MgSO.sub.4 and concentrated under reduced pressure. The
resulting oil was subjected to flash chromatography in 30% ethyl
acetate/hexanes, which yielded-diketo alcohol 13 (0.015 g, 0.051
mmol, 70%) as a colorless oil. ##STR103##
[0415] To a stirred solution of 13 (1.28 g, 2.02 mmol) in toluene
(30 ml) was added p-toluene sulfonic acid (0.060 g, 0.0003 mmol) in
a single portion followed by addition of 4 .ANG. molecular sieves
(1.5 g). After stirring at rt for 10 hours, the reaction was
quenched with pyridine (2.0 ml, 24.7 mmol) and filtered. The
resulting solution was concentrated under reduced pressure, then
redissolved in dietyhl ether. This was washed with saturated
NaHCO.sub.3 and dried over MgSO.sub.4 before the solvent was
removed under reduced pressure. The resulting oil was subjected to
flash chromatography in 30% ethyl acetate in hexanes, which was
increased to 70% as the product began to elute from the column.
This yielded 1.00 g (1.63 mmol, 81%) of 14 as a 1.54:1 mixture of
diastereomers at C23 which were separable after a second
chromatographic step in which 300 g of silica gel were used per 1 g
of the pure diastereomers, and the material was eluted again using
30% ethyl acetate in hexanes. ##STR104##
[0416] In a glove bag, under positive Ar pressure, 0.9834 g (3.11
mmole) methoxy diisopinylborane was weighed into a dried flask. Dry
diethyl ether (8.4 ml) was added, and the resulting solution cooled
to -78.degree. C. To this solution was added dropwise over 5
minutes a 1M solution of allyl magnesium bromide (2.8 ml, 2.8
mmol), after which the solution was allowed to come to rt over 1
hour. A portion (6.2 ml, 2.06 mmole) of the resulting borane
reagent was added to a stirred solution of the aldehyde 10 (0.6546
g, 2.0 mmol) in diethyl ether (5 ml) at -78.degree. C. over 15
minutes. The reaction was stirred at -78.degree. C. for 1 hour, and
then allowed to warm to rt over 1 hour. The resulting boronate was
then cleaved by addition of 10 ml of 15% NaOH and 2 ml 30%
H.sub.2O.sub.2. This mixture was stirred for 30 minutes, and the
layers were separated. The aqueous layer was then extracted 4 times
with diethyl ether. The combined organic phases were dried over
MgSO.sub.4 and concentrated under reduced pressure. The resultant
oil was subjected to flash chromatography in 15% ethyl
acetate/hexanes produced 15 (0.4723 g, 1.28 mmol, 64%) as a clear
oil. ##STR105##
[0417] To a stirred solution of 15 (3.39 g, 9.19 mmol) in dry
CH.sub.2Cl.sub.2 (80 ml) was added a 60% dispersion of NaH in
mineral oil (0.551 g, 13.79 mmol) in a single portion, and the
resulting solution was stirred at rt for 1 h. To this was added
dimethyl aminopyridine (0.061 g, 0.5 mmol), followed by t-butyl
dimethylsilyl chloride (2.216 g, 14.70 mmol). The solution was then
refluxed for 16 h, after which the solution was cooled to rt and
quenched with 10 ml of a saturated solution of aqueous ammonium
chloride. The layers were separated, and the aqueous phase
extracted 3 times with ethyl acetate. The combined organic phases
were dried over MgSO.sub.4 and concentrated under reduced pressure.
The resultant oil was subjected to flash chromatography in 7.5%
ethyl acetate/hexanes, yielding the TBS protected allyl alcohol 27
(3.833 g, 7.90 mmol, 86%) as a clear oil. ##STR106##
[0418] To a stirred solution of the previously prepared TBS allyl
alcohol 27 (0.035 g, 0.072 mmol) in 2:1 THF/water (3 ml) was added
N-methyl morpholine N-oxide (0.0092 g, 0.079 mmol) in one portion,
followed by the rapid addition of a 2.5% solution of osmium
tetroxide in isopropanol (0.090 ml, 0.07 mmol), again in one
portion. The resultant solution was stirred 6 hours at rt before
being stopped by the addition of excess solid sodium sulfite. The
reaction mixture was then diluted with brine and ethyl acetate. The
layers were separated, and the aqueous phase extracted three times
with ethyl acetate. The combined organic phases were concentrated
under reduced pressure. The resultant oil was redissolved in 3 ml
2:1 tetrahydrofuran/water, to which was added sodium periodate
(0.052 g, 0.243 mmol). The reaction mixture was stirred 6 hours at
rt before being diluted with water and ethyl acetate. The layers
were separated, and the aqueous phase extracted three times with
ethyl acetate, and the combined organic phases concentrated under
reduced pressure. The resultant yellow oil was subjected to flash
chromatography in 10% ethyl acetate/hexanes, and yielded aldehyde
16 (0.0297 g, 0.061 mmol, 85% over 2 steps) as a clear oil.
##STR107##
[0419] To a stirred solution of diisopropyl amine (0.352 ml, 2.51
mmol) in dry THF (25 ml) at -78.degree. C. was added dropwise a
2.5M solution of nBuLi in hexanes (0.956 ml, 2.39 mmol). The
solution was stirred for 30 minutes at 0.degree. C. and then cooled
to -78.degree. C. A solution of methyl ketone 9 (0.592 g, 2.26
mmol) in dry THF (5 ml) was cannulated slowly into the reaction
mixture over 10 minutes. The resulting solution was stirred at
-78.degree. C. for 30 minutes, warmed to rt for 2 minutes, then
recooled to -78.degree. C., after which a solution of aldehyde 16
(1.0 g, 2.05 mmol) in dry THF (3 ml) was slowly cannulated into the
reaction mixture over 10 minutes. This was stirred at -78.degree.
C. for 15 minutes, then quenched with a saturated solution of
aqueous ammonium chloride (25 ml). The reaction was then diluted
with ethyl acetate, and the layers separated. The aqueous phase was
extracted 3 times with ethyl acetate, and the combined organic
phases dried with MgSO.sub.4 and concentrated under reduced
pressure. The resulting oil was subjected to flash chromatography
in 10% ethyl acetate in hexanes, which was increased to 20% as the
product began to elute from the column. This yielded 1.48 g (1.97
mmol, 96%) of an inconsequential mixture of diastereomers which
were carried through to the next reaction.
[0420] To a stirred solution of the diastereomeric mixture of
.beta.-keto alcohols (0.1103 g, 0.147 mmol) in dry CH.sub.2Cl.sub.2
was added N-methyl morpholine N-oxide (0.1418 g, 1.207 mmol) and
approximately 0.1 g powdered 4 .ANG. molecular sieves. Tetrapropyl
ammonium perruthenate (0.0131 g, 0.037 mmol) was added and the
mixture stirred for 30 minutes. The reaction mixture was then
filtered through a short plug of silica with copious quantities of
ethyl acetate. The resulting solution was concentrated under
reduced pressure and subjected to flash chromatography using 15%
ethyl acetate in hexanes. This yielded 0.0513 g (0.069 mmol, 47%)
of the desired .beta.-diketone 28. ##STR108##
[0421] To a solution of pyranone 14 (0.205 g, 0.333 mmol) and
cerium chloride heptahydrate (0.030 g, 0.082 mmol) in 5.5 ml
methanol was added solid NaBH.sub.4 (0.012 g, 0.33 mmol) in a
single portion at -20.degree. C. The reaction mixture was stirred
for 1 hour at -20.degree. C. and monitored by tlc. A second portion
of NaBH.sub.4 (0.012 g, 0.33 mmol) was added during this period
when the reaction appeared stalled. The reaction was then quenched
with 20 ml saturated aqueous NaCl, and the mixture brought to rt,
filtered through a pad of Celite.RTM. and the layers separated. The
separated aqueous layer was extracted four times with ethyl
acetate, and the combined organics were dried over Na.sub.2SO.sub.4
and concentrated under reduced pressure to afford the crude allylic
alcohol. This moderately stable oil was reacted further without
purification.
[0422] The crude allylic alcohol was dissolved in 6 ml
CH.sub.2Cl.sub.2/MeOH (2:1) and cooled to 0.degree. C. before being
treated with solid NaHCO.sub.3 (0.042 g, 0.5 mmol). Purified
m-chloroperoxybenzoic acid (0.046 g, 0.370 mmol) was added in a
single portion and the reaction mixture was stirred for 30 minutes,
then warmed to rt over a period of 15 minutes. The reaction was
quenched with triethylamine (4.0 ml), stirred well for 20 minutes,
diluted with 40 ml diethyl ether, and filtered through a pad of
Celite.RTM.. The filtrate was concentrated under reduced pressure
and the resulting oil purified using flash chromatography using
(EtOAc/hexane 1/1) to give diol 17 (0.158 g, 0.238 mmol, 71%) as a
colorless oil. ##STR109##
[0423] A solution of diol 17 (118 mg, 0.178 mmol) and
4-dimethylaminopyridine (77 mg, 0.62 mmol) in CH.sub.2Cl.sub.2 (3.2
ml) was cooled to -10.degree. C. and treated with benzoyl chloride
(27 .mu.l, 0.23 mmol) dropwise via syringe. The resulting mixture
was stirred at -10.degree. C. for 30 minutes, quenched with
saturated NaHCO.sub.3 and diluted with EtOAc (20 ml). The organic
layer was washed with H.sub.2O and brine, dried over
Na.sub.2SO.sub.4 and concentrated in vacuo to afford a crude
mixture of C21 monobenzoate and 4-dimethylaminopyridine as a
colorless paste, which was filtered over a plug of silica gel
(EtOAc/hexane 1/2).
[0424] The filtrate was evaporated and taken up in 8 ml
CH.sub.2Cl.sub.2 and treated with solid
1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (Dess-Martin
periodinane, 113 mg, 0.267 mmol) at rt. The solution was stirred
for 4 hours at rt after which a second portion (113 mg, 0.267 mmol)
of DMP was added. The opaque white mixture was stirred for another
1.5 hours and quenched with 4 ml saturated
NaHCO.sub.3/Na.sub.2S.sub.2O.sub.3. The layers were separated and
the aqueous phase was extracted with CH.sub.2Cl.sub.2 (2.times.).
The combined organics were dried over Na.sub.2SO.sub.4 and
concentrated in vacuo to provide a colorless semisolid. Flash
chromatography on silica gel (EtOAc/hexane 1/3) gave the desired
keto-pyranone 29 (121 mg, 89% from 17) as a colorless oil.
##STR110##
[0425] To a stirred solution of diisopropylamine (300 .mu.l, 2.14
mmol) in THF (3.2 ml) was added n-butyllithium (1.25 ml, 1.6 M in
hexanes, 2.00 mmol) dropwise at -78.degree. C. The mixture was
warmed to 0.degree. C. and stirred for 30 min, then cooled again to
-78.degree. C., and a solution of ketone 18 (319 mg, 0.494 mmol) in
6.8 ml THF was added in a single portion. The solution was stirred
for 30 minutes and treated with a solution of freshly distilled
OHCCO.sub.2Me (88 mg, 0.74 mmol) in 5 ml THF, kept at -78.degree.
C. for 30 minutes and quenched with 3 ml saturated NH.sub.4Cl. The
reaction mixture was brought to rt and diluted with 200 ml EtOAc.
The organic layer was washed with H.sub.2O (2.times.) and brine,
dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude
residue was chromatographed on silica gel (EtOAc/hexanes 35/65) to
afford the aldol product (319 mg, 88%) as an inconsequential
mixture of diastereomers.
[0426] The isolated aldol product (303 mg, 0.412 mmol) and
Et.sub.3N (340 .mu.l, 2.50 mmol) were dissolved in anhydrous
CH.sub.2Cl.sub.2 (15 ml) and cooled to -10.degree. C.
Methanesulfonylchloride (97 .mu.l, 1.25 mmol) was added via syringe
and the solution was stirred at -10.degree. C. for 30 minutes and
warmed to rt. 5 ml saturated NaHCO.sub.3 were added and the
reaction mixture was diluted with 100 ml EtOAc. The organic layer
was washed with H.sub.2O and brine, dried over Na.sub.2SO.sub.4 and
concentrated in vacuo. The residue was immediately dissolved in THF
(20 ml) and treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU-75
.mu.A, 0.5 mmol) dropwise at rt. The resulting bright yellow
solution was stirred at rt for 20 minutes, treated with saturated
NH.sub.4Cl and diluted with 150 ml EtOAc. The organic layer was
washed with H.sub.2O and brine, dried over Na.sub.2SO.sub.4 and
concentrated in vacuo to afford an orange residue which was
chromatographed on silica gel (20% EtOAc/hexanes) to afford
exocyclic methacrylate 30 (239 mg, 81%--unseparable mixture of E/Z
isomers, ratio E/Z=7:1) as a yellow oil. ##STR111##
[0427] To a solution of enone 19 (205 mg, 0.286 mmol) and cerium
chloride heptahydrate (52 mg, 0.15 mmol) in 11 ml methanol was
added solid NaBH.sub.4 (21 mg, 0.57 mmol) in a single portion at
-30.degree. C. Rapid gas evolution subsided after 3 minutes. After
an additional 30 minutes at -30.degree. C., the reaction mixture
was poured directly onto a silica gel column and the product
quickly eluted with EtOAc/hexanes (3/1) to afford the alcohol as
colorless oil.
[0428] Octanoic acid (93 mg, 0.64 mmol) and Et.sub.3N (117 .mu.l,
0.88 mmol) were dissolved in 8 ml toluene and treated with
2,4,6-trichlorobenzoylchloride (92 .mu.l, 0.59 mmol) dropwise at
rt. After 2.5 hours at rt, a toluene solution (5 ml) of freshly
prepared alcohol was added gradually via syringe and stirring was
continued for 1 h. The reaction mixture was quenched with 10 ml
saturated NaHCO.sub.3, diluted with EtOAc and washed successively
with saturated NH.sub.4Cl and brine. The organics were dried over
Na.sub.2SO.sub.4, the solvent was removed in vacuo, and the residue
was chromatographed on silica gel (EtOAc/hexane 1/3) to provide
octanoate 20 as an colorless oil (208 mg, 86% from 19).
##STR112##
[0429] To a solution of 20 (2.0 mg, 0.0024 mmol) in 1 mL 1% aqueous
CH.sub.2Cl.sub.2 was added solid
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 1.2 mg, 0.0053
mmol) at 0.degree. C. The reaction mixture was stirred for 30
minutes, then pipetted directly onto a plug of silica gel. The
product was eluted with EtOAc/hexane (1/4) and the solvent was
removed in vacuo to provide the crude diol (1.1 mg). This material
was dissolved in anhydrous CH.sub.2Cl.sub.2 and treated with
manganese (IV) oxide (0.4 mg, 0.0046 mmol) at 0.degree. C. The
reaction mixture was allowed to warm to rt and then pipetted
directly onto a plug of silica gel. The product was eluted with
EtOAc/hexane (1/3) and the solvent was removed in vacuo. Crude
aldehyde 33 (0.64 mg, 42%) was obtained as a colorless oil.
Example 12
[0430] Compound and synthetic step references in this example
correspond to the following scheme (as opposed to the reaction
schemes and formula numbers employed previously in the
specification). ##STR113## ##STR114##
[0431] A 3-neck flask was charged with NaH (16 g, 60 wt % in
mineral oil, 0.40 mol). The NaH was washed with Et.sub.2O
(3.times.40 mL). THF (800 mL) was added and the mixture stirred. To
this suspension was added 2,2-dimethyl-1,3-propanediol 1 (40 g,
0.39 mol) in protions over 10 min. The resulting thick slurry was
stirred at room temperature [rt] for 1 h. TBSCl (60.5 g, 0.40 mol)
was added in one portion. The slurry thinned and was stirred at rt
for 14 h. The reaction was diluted with MTBE (1.0 L) and washed
with 10% aq. K.sub.2CO.sub.3 (700 mL, 300 mL) and brine (500 mL).
The organic layer was dried over Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure to yield 88.4 g of a viscous
liquid which was used without further purification. ##STR115##
[0432] A solution of 2 (40 g, ca 0.18 mol) was stirred in
CH.sub.2Cl.sub.2 (900 mL) and cooled in an ice bath to 4.degree. C.
To this solution was added NEt.sub.3 (76 mL, 0.54 mol). A slurry of
SO.sub.3 pyr (44 g, 0.27 mol) in DMSO (100 mL) was added in two
portions 5 min apart to keep T<10.degree. C. The reaction was
stirred for 2.5 h and the ice bath removed. Stirring was continued
for 6.5 h and another portion of SO.sub.3 pyr (10 g, 63 mmol) was
added as a solid. The reaction was stirred for 9 h and diluted with
CH.sub.2Cl.sub.2 (1.0 L). The resulting solution was washed with 1
N aq. HCl (2.times.500 mL), satd. aq. NaHCO.sub.3 (500 mL), and
brine (500 mL). The resulting organic layer was dried over
Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure
to 39.8 g of an orange liquid which was used without further
purification. ##STR116##
[0433] A solution of 4-chloro-1-butanol (23 mL, 0.23 mol) in THF
(230 mL) was cooled to -78.degree. C. A solution of MeMgCl (77 mL,
3.0 M in THF, 0.23 mol) was added dropwise via addition funnel over
30 min to keep T<-60.degree. C. The reaction was let warm to
-10.degree. C. and Mg.sup.0 (6.04 g, 0.251 mol) was added followed
by BrCH.sub.2CH.sub.2Br (0.1 mL). The reaction was heated to reflux
for 14.5 h. Heating was removed, THF (220 mL) was added, and the
reaction was cooled to -78.degree. C. A solution of 3 (39 g, ca
0.18 mol) in THF (100 mL) was added dropwise via addition funnel
over 40 min to keep T<-60.degree. C. The reaction was stirred 30
min at -78.degree. C. and the cold bath removed. MTBE (500 mL) was
added when the reaction reached -30.degree. C. followed by aq.
citric acid (87 g, 0.41 mol in 500 mL H.sub.2O). The organic layer
was collected and the aqueous layer was extracted with MTBE (200
mL). The combined organic layers were washed with brine
(2.times.400 mL), dried over Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure to a light orange oil. The oil
was dissolved in EtOAc and filtered through silica. The filtrate
was concentrated under reduced pressure to yield 49.7 g of a light
yellow oil which was used without further purification.
##STR117##
[0434] To a solution of CH.sub.2Cl.sub.2 (800 mL) was added
(COCl).sub.2 (46.9 mL, 0.537 mol) and the resulting solution cooled
to -78.degree. C. DMSO (75.8 mL, 1.07 mol) was added dropwise via
addition funnel over 15 min to keep T<-60.degree. C. The
resulting solution was stirred for 10 min. A solution of 4 (49.7 g,
ca 0.179 mol) in CH.sub.2Cl.sub.2 (150 mL) was added dropwise via
addition funnel over 25 min to keep T<-70 C. The resulting
mixture was stirred 1 h at -78.degree. C. and NEt.sub.3 (250 mL,
1.79 mol) was added dropwise via addition funnel over 15 min to
keep T<-60.degree. C. The cold bath was removed and the reaction
was allowed to warm to -30.degree. C. and then poured into a
mixture of H.sub.2O (500 mL) and CH.sub.2Cl.sub.2 (800 mL). The
resulting organic layer was collected and washed with H.sub.2O (500
mL), satd. aq. NH.sub.4Cl (2.times.400 mL), satd. aq. NaHCO.sub.3
(500 mL), and brine (500 mL). The organic layer was then dried over
Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure
to a dark oil. Purification by flask chromatography on silica in
19:1.fwdarw.9:1 pet. ether:EtOAc yielded 27.81 g (54% based on
2,2-dimethyl-1,3-propanediol) of a straw yellow liquid.
##STR118##
[0435] A 3-neck flask was charged with powdered 4 .ANG. mol. sieves
(47.5 g), CH.sub.2Cl.sub.2 (600 mL), and R-BINOL (3.6 g, 12.5
mmol). To this mixture was added Ti(OiPr).sub.4 (1.84 mL, 6.25
mmol). The resulting orange mixture was heated to reflux for 1 h,
then cooled to rt in a water bath. A solution of 5 (36.0 g, 125
mmol) in CH.sub.2Cl.sub.2 (60 mL) was added followed by
B(OMe).sub.3 (16.8 mL, 150 mmol) and Bu.sub.3SnCH.sub.2CHCH.sub.2
(46.5 mL, 150 mmol). The resulting mixture was stirred at rt for 42
h, then filtered through celite into saturated aqueous NaHCO.sub.3
(200 mL). The resulting mixture was stirred for 1 h. The organic
layer was collected and the aqueous layer was extracted with
CH.sub.2Cl.sub.2 (200 mL). The combined organic layers were dried
over Na.sub.2SO.sub.4, filtered, and concentrated under reduced
pressure to a thick orange oil. The residue was purified by flash
chromatography on silica in 9:1.fwdarw.4:1 pet. ether:EtOAc to
yield 29.03 g, (70%) of a yellow oil. ##STR119##
[0436] To a solution of 6 (28.8 g, 87.6 mmol) in MePh (500 mL) was
added beaded 4 .ANG. mol. sieves (50 g) and pTsOH (1.66 g, 8.76
mmol). The mixture was stirred at rt for 18 h then filtered through
basic alumina (Brockman grade I, basic, 150 mesh) and the alumina
washed with pet. ether. The filtrate was concentrated under reduced
pressure to 23.2 g (85%) of a clear liquid. ##STR120##
[0437] A mixture of 80% MMPP (25.7 g, 41.5 mmol), CH.sub.2Cl.sub.2
(245 mL), MeOH (122 mL), and NaHCO.sub.3 (8.87 g) was stirred in an
ice bath. A solution of 7 (21.5 g, 69.2 mmol) in CH.sub.2Cl.sub.2
(50 mL) was added dropwise via addition funnel over 10 min. The
reaction was stirred for 10 min, and then poured into H.sub.2O (200
mL). The organic layer was collected and the aqueous layer was
extracted with CH.sub.2Cl.sub.2 (200 mL). The combined organic
layers were dried over Na.sub.2SO.sub.4, filtered, and concentrated
under reduced pressure to a thick oil which was purified by flash
chromatography on silica in 19:1.fwdarw.9:1 pet. ether:EtOAc to
yield 16.49 g (66%) of a clear oil. ##STR121##
[0438] To an oven-dried flask was added the major diastereomer of 8
(5.3 g, 14.8 mmol) in 75 mL CH.sub.2Cl.sub.2. MeCN (12 mL) was
added and the reaction was cooled in an ice bath. 7.6 g 4 .ANG.
molecular sieves was added, followed by NMO (2.6 g, 22.2 mmol) and
TPAP (310 mg, 0.88 mmol). The reaction was stirred for 15 min. in
an ice bath, then warmed to rt. Reaction stirred overnight.
Additional 4 .ANG. molecular sieves (1.05 g), NMO (1.0 g), and TPAP
(120 mg) were added. After 5 hours of stirring, at rt, the reaction
was filtered through celite, with a CH.sub.2Cl.sub.2 was. The
filtrate was concentrated in vacuo and flashed through a plug of
silica, eluting with CH.sub.2Cl.sub.2. The fractions containing
product were concentrated in vacuo to yield 4.28 g (81%) of
product. ##STR122##
[0439] To a solution of 9 (1.72 g, 4.82 mmol) in MeOH (50 mL) was
added K.sub.2CO.sub.3 (3.66 g, 26.5 mmol) and a solution of methyl
glyoxylate (14.2 mL, .about.1.7 M, 24 mmol) in THF. The resulting
mixture was stirred at rt for 55 min and then poured into a mixture
of satd. aq. NH.sub.4Cl (200 mL) and Et.sub.2O (100 mL). The
organic layer was collected and the aqueous layer was extracted
with Et.sub.2O (100 mL). The combined organic layers were dried
over MgSO.sub.4, filtered, and concentrated under reduced pressure
to an orange oil which was purified by flash chromatography on
silica in 19:1 pet. ether:EtOAc to yield 1.50 g (72%) of a yellow
oil which solidified on standing. ##STR123##
[0440] To an oven-dried flask was added 10 (3.29 g, 7.71 mmol) in
MeOH (130 mL). CeCl.sub.3.7H.sub.2O (1.44 g, 3.86 mmol) was added
and the reaction was stirred until the salts dissolved. The
reaction was then cooled to -30.degree. C., and NaBH.sub.4 (580 mg,
15 mmol) was added. The reaction was stirred for 20 min at
-30.degree. C. The reaction was loaded directly onto a silica
column (250 g) and eluted with 6:1 hexanes:EtOAc. The fractions
containing product were combined and washed with H.sub.2O
(2.times.85 mL) and brine (3.times.85 mL). The organic layer was
then dried over Na.sub.2SO.sub.4, filtered, and concentrated in
vacuo to yield 3.49 g of crude product which was used directly in
the next reaction.
[0441] The crude alcohol was dissolved in CH.sub.2Cl.sub.2 (75 mL)
and DMAP (1.41 g, 11.5 mmol) was added. Octanoic acid (1.83 mL,
11.5 mmol) was then added, followed by DIC (1.81 mL, 11.6 mmol).
The reaction was stirred at rt for 20 h. The reaction was then
diluted with EtOAc and brine. The organic layer was collected and
the aqueous layer was extracted with EtOAc (1.times.). The combined
organic layers were washed with brine, dried over Na.sub.2SO.sub.4,
filtered, and concentrated to 7.17 g of a solid/oil mixture. The
crude material was purified via column chromatography (silica (300
g), hexanes:EtOAc, 19:1) to yield 3.98 g (93% over 2 steps) of pure
product as a light yellow oil. ##STR124##
[0442] To a solution of TBS ether 12 (2.0 g, 3.61 mmol) in THF
(36.1 mL) at rt was added 3.HF.Et.sub.3N (6.0 mL, 36.1 mmol). The
reaction mixture was stirred for 48 h and then diluted with ether
(36 mL). The organic phase was washed with sat. aq. NaHCO.sub.3
(2.times.20 mL) and brine (2.times.20 mL), dried over
Na.sub.2SO.sub.4, and flashed through a plug of silica. The residue
obtained after evaporation of solvent was carried forward without
further purification. R.sub.f=0.20 (hexane/ethyl acetate 4:1).
[0443] To a solution of the deprotected alcohol in CH.sub.2Cl.sub.2
(21 mL) and MeCN (2.1 mL), at rt, was added 4 .ANG. molecular
sieves (powder, 1.81 g) and NMO (1.06 g, 9.03 mmol). The mixture
was cooled to 0.degree. C. and TPAP (127 mg, 0.361 mmol) was added
in one portion. The reaction mixture was stirred for 10 min.
0.degree. C. and for 2 hrs. at rt. The reaction was filtered
through celite, eluting with CH.sub.2Cl.sub.2, and concentrated in
vacuo. The crude reaction mixture was then flashed through a plug
of silica, also eluting with CH.sub.2Cl.sub.2, and then
concentrated in vacuo. Purification via column chromatography
(silica gel, 2.5% EtOAc/Petroleum Ether) revealed pure aldehyde 14
(1.20 g, 76% over 2 steps) as a yellow oil. ##STR125##
[0444] 1-bromo-2-ethoxyethylene (690 .mu.L, 6.5 mmol) was added to
an oven-dried flask containing Et.sub.2O (22 mL). The solution was
cooled to -78.degree. C., and t-BuLi (7.63 mL, 13 mmol, 1.7M in
pentane) was added, dropwise. The reaction was stirred, at
-78.degree. C., for 30 min. Me.sub.2Zn (3.35 mL, 6.7 mmol, 2.0M in
toluene) was added, dropwise, and the reaction was stirred, at
-78.degree. C., for 30 min. A solution of aldehyde 14 (0.948 g, 2.2
mmol dissolved in Et.sub.2O (22 mL, 0.1 mmol) was added dropwise,
via syringe, and the reaction was stirred for 2 h at -78.degree. C.
The reaction was quenched with a 1.0 M solution of HCl (40 mL) and
allowed to warm to rt. The mixture stirred vigorously for 19 h and
was quenched with sat'd aq. NaHCO.sub.3 (60 mL), diluted with EtOAc
(35 mL) and H.sub.2O (35 mL). The separated aqueous phase was
extracted with EtOAc (3.times.60 mL) and the combined organic
phases washed with brine (1.times.85 mL). The organic layer was
dried (Na.sub.2SO.sub.4), filtered, and concentrated in vacuo.
Purification via column chromatography (silica gel, Pet
Ether.fwdarw.7%.fwdarw.10% EtOAc:Petroleum Ether) revealed pure
enal 16 (905 mg, 90%) as a nearly colorless oil. ##STR126##
[0445] Preparation of stock solution: K.sub.2OsO.sub.2(OH).sub.4
(2.0 mg, 0.0054 mmol), (DHQD).sub.2PYR (12.0 mg, 0.0136 mmol),
K.sub.3Fe(CN).sub.6 (1.34 g, 4.07 mmol), and K.sub.2CO.sub.3 (563
mg, 4.07 mmol) were combined in a round-bottom flask, to which was
added 6.75 mL of H.sub.2O and 6.75 mL of tBuOH. The two-phase
system was vigorously stirred at rt for 2 h.
[0446] Enal 16 (300 mg, 0.646 mmol) was cooled to 0.degree. C., and
a 6.4 mL aliquot of the stock solution was added. The yellow-orange
colored reaction mixture was stirred at 0-4.degree. C. for 60 h (in
cold room). After diluting with water (50 mL) the aqueous phase was
extracted with ethyl acetate (3.times.250 mL). The combined organic
phases were dried over MgSO.sub.4, and then filtered. The solvents
were removed in vacuo. Purification via column chromatography
(silica gel, hexane/ethyl acetate 1:9) yielded diol 17 (233 mg,
72%) as a 2.5:1 inseparable mixture of diastereomers.
##STR127##
[0447] The diol (10 mg, 0.020 mmol) was taken up in a solution of
MeCN:H.sub.2O (960 .mu.L, 240 .mu.L), and transferred to an
oven-dried flask. pTsOH (38 mg, 0.200 mmol) was added as 0.22M
solution in MeCN:H.sub.2O (730 .mu.L, 180 .mu.L). The reaction was
stirred overnight, at rt, and then quenched with sat. aq.
NaHCO.sub.3 (.about.6 mL). The aqueous phase was extracted with
EtOAc (.times.3). The combined organic layers were dried over
Na.sub.2SO.sub.4, filtered, and concentrated in vacuo. The crude
product was carried forward without further purification.
[0448] A 0.5M stock solution of 1:3 TBSCl:imidazole was prepared by
dissolving TBSCl (754 mg, 5.0 mmol) and imidazole (1.02 g, 15.0
mmol) in CH.sub.2Cl.sub.2 (10.0 mL). A total of 9 eq. of this stock
solution were added to the reaction over 6 hrs. (added in 3 eq.
aliquots, 2 hrs. apart). Reaction was quenched with sat. aq.
NH.sub.4Cl and the aqueous phase was extracted with EtOAc (.times.3
mL). The combined organic phases were dried over Na.sub.2SO.sub.4,
filtered, and concentrated in vacuo. Purification via column
chromatography (silica gel, Hexanes:EtOAc, 75:25) revealed pure
product (5.6 mg, 47% over 2 steps).
Example 13
[0449] Compound and synthetic step references in this example
correspond to the following scheme (as opposed to the reaction
schemes and formula numbers employed previously in the
specification). ##STR128##
[0450] To a solution of acid 31 (28 mg, 0.05 mmol) in toluene (1.1
mL) was added NEt.sub.3 (14 mL, 0.1 mmol) and
2,4,6-trichlorobenzoyl chloride (8.3 .mu.L, 0.05 mmol) at rt and
the mixture was stirred for 1 h. To this reaction mixture was added
a solution of the alcohol 19 (15 mg, 0.025 mmol) and DMAP (15 mg,
0.125 mmol) in toluene (1.1 mL) at rt. The reaction mixture was
stirred for 1 h and then directly poured onto a column (silica gel,
hexane/ethyl acetate 85:15) and diluted with this solvent mixture
to afford 24 mg (87%) of ester 32 as a colorless oil.
##STR129##
[0451] To a solution of aldehyde 32 (20 mg, 0.018 mmol) in THF (5
mL) at rt in a plastic vial was added 70% HF/pyridine (968 .mu.L,
excess) dropwise and the yellow reaction mixture was stirred for
1.5 h. The reaction was quenched by addition of sat. aq.
NaHCO.sub.3 solution (12.5 mL) and H.sub.2O (7.5 mL). The biphasic
mixture was extracted with ethyl acetate (4.times.12 mL). The
combined organic phases werde dried over Na.sub.2SO.sub.4. After
filtration and evacuation of solvents the residue was purified by
column chromatography (silica gel, hexane/ethyl acetate 1:4) to
yield 11 mg (82%) of Formula 33 as a white solid.
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[0497] All references cited herein are hereby incorporated by
reference. Although the invention has been described with respect
to specific embodiments and examples, it will be appreciated that
various changes and modifications may be made without departing
from the spirit of the invention.
* * * * *