U.S. patent application number 10/214653 was filed with the patent office on 2003-05-22 for synthesis of oligoketides.
Invention is credited to Ashley, Gary, Burlingame, Mark A., Chan-Kai, Isaac C..
Application Number | 20030096374 10/214653 |
Document ID | / |
Family ID | 22372623 |
Filed Date | 2003-05-22 |
United States Patent
Application |
20030096374 |
Kind Code |
A1 |
Ashley, Gary ; et
al. |
May 22, 2003 |
Synthesis of oligoketides
Abstract
Facile methods for preparing diketide and triketide thioesters
are disclosed. The resulting thioesters may be used as
intermediates in the synthesis of desired polyketides, and may
contain functional groups which ultimately reside in side chains on
the resulting polyketide and thus can be used further to manipulate
the polyketide so as form derivatives. The polyketides produced may
also be tailored by glycosylation, hydroxylation and the like. New
polyketides and their derivatives and tailored forms are thereby
produced.
Inventors: |
Ashley, Gary; (Alameda,
CA) ; Chan-Kai, Isaac C.; (Hayward, CA) ;
Burlingame, Mark A.; (San Francisco, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
22372623 |
Appl. No.: |
10/214653 |
Filed: |
August 7, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10214653 |
Aug 7, 2002 |
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09492733 |
Jan 27, 2000 |
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6492562 |
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60117384 |
Jan 27, 1999 |
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Current U.S.
Class: |
435/74 ;
536/7.4 |
Current CPC
Class: |
C07C 327/30
20130101 |
Class at
Publication: |
435/74 ;
536/7.4 |
International
Class: |
C12P 019/44; C07H
017/08 |
Claims
1. A method to prepare a diketide or triketide thioester which
method comprises a) treating a benzoxazolone derivative of said
diketide or triketide with the salt of a thiol anion from which the
thioester is to be formed so as to form the thioester of said
diketide or triketide; or b) treating a 2-oxazolidinone derivative
of said diketide or triketide with the lithium salt of a thiol
anion from which the thioester is to be formed in the presence of
sufficient Lewis acid to reduce the basicity of the thiol anion so
as to form the thioester of said diketide or triketide.
2. The method of claim 1 wherein said thioester is an N-acyl
cysteamine thioester.
3. The method of claim 1 wherein the Lewis acid is
trimethylammonium.
4. A diketide or triketide thioester prepared by the method of any
of claims 1-3.
5. A method to prepare a polyketide which method comprises treating
a polyketide synthase (PKS) enzyme complex with the diketide or
polyketide thioester of claim 4 under conditions wherein said
polyketide is formed.
6. The method of claim 5 wherein said PKS is contained in a
cell.
7. The method of claim 5 wherein said polyketide has the lactone
backbone structure of 6-dEB.
8. A polyketide prepared by the method of any of claims 5-7.
9. The polyketide of claim 8 which contains a functional group in a
side chain at position 13 of the lactone.
10. The polyketide of claim 9 wherein the functional group is a
double bond, a triple bond, a halo group, an azide, an ester, an
alcohol, or an aromatic nucleus.
11. The polyketide of claim 10 wherein the functional group is a
double bond, a halo group, an azide, or an aromatic nucleus.
12. The polyketide of claim 8 which contains a functional group at
a side chain coupled to the 12 position of the lactone.
13. The polyketide of claim 12 wherein said functional group is a
double bond.
14. A method to prepare a tailored polyketide which method
comprises treating the polyketide of claim 8 with tailoring
enzymes.
15. The method of claim 14 wherein the tailoring enzymes are
contained in a cell.
16. A tailored polyketide prepared by the method of claim 15.
17. The tailored polyketide of claim 16 which comprises
hydroxylation or glycosylation.
18. A method to prepare a derivatized polyketide or tailored
polyketide which method comprises contacting the polyketide of any
of claims 9-13 or tailored polyketide of claim 16 or 17 with a
suitable reagent compatible with said functional group.
19. A derivatized polyketide or tailored polyketide prepared by the
method of claim 18.
20. The method of claim 1 wherein said diketide or triketide
comprises 2-methyl-3-hydroxy substituents.
21. The method of claim 20 wherein said diketide or triketide
substituents have syn chirality.
22. The method of claim 21 wherein said diketide or triketide is
selected from the group consisting of 2-methyl-3-hydroxyhexanoyl;
2-methyl-3-hydroxy-4-pentenoyl; 2-methyl-3-hydroxybutanoyl;
2-vinyl-3-hydroxypentanoyl; and
2,4-dimethyl-3,5-dihydroxyheptanoyl.
23. The method of claim 1 wherein said diketide or triketide is
selected from the group consisting of
(2S,3R)-2-methyl-3-hydroxyhexanoyl;
(2S,3R)-2-methyl-3-hydroxy-4-pentenoyl;
(2S,3R)-2-methyl-3-hydroxybutanoy- l;
(2S,3R)-2-vinyl-3-hydroxypentanoyl;
(2S,4S,5R)-2,4-dimethyl-5-hydroxy-3- -oxoheptanoyl;
(2S,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl; and
(4S,5R)-4-methyl-5-hydroxy-2-heptenoyl.
24. A method to synthesize a derivative of at least a triketide
containing stereochemically pure chiral centers at at least
positions 2 and 5 which method comprises treating a
stereochemically controlled diketide derivative having a chiral
center at position 2 of said diketide with an aldehyde in the
presence of tin(II) triflate and titanium tetrachloride so as to
maintain the chirality at position 2 and provide control of the
chirality at position 5.
25. The method of claim 24 wherein said sterically controlled
diketide is a derivative of 2-oxazolidinone.
26. A method to synthesize an oligoketide thioester on a solid
support, which method comprises (1) reacting an
N-acyl-2-imidazolidinone coupled to said solid support with an
aldehyde or acyl moiety under conditions whereby said aldehyde or
acyl moiety couples to a position .alpha. to a carbonyl in the acyl
group of the 2-imidazolidinone; (2) optionally repeating step (1);
and (3) cleaving the resulting oligoketide from the solid support
by reaction with a salt of a thiol anion, thus providing an
oligoketide thioester.
27. The method of claim 26 wherein the salt is a lithium salt,
and/or wherein said cleaving is performed in the presence of a
Lewis acid.
28. A method to synthesize an oligoketide thioester on a solid
support, which method comprises (1) reacting an N-acyl
benzoxazolone coupled to said solid support with an aldehyde under
conditions whereby said aldehyde couples to a position .alpha. to a
carbonyl in the acyl group of the benzoxalozone; (2) optionally
repeating step (1); and (3) cleaving the resulting oligoketide from
the solid support by reaction with a salt of a thiol anion, thus
providing an oligoketide thioester.
29. A method to synthesize a racemic mixture of diketides which
method comprises treating an N-acyl derivative of a benzoxazolone
with an aldehyde under conditions wherein said aldehyde couples to
a position alpha to the carbonyl in the acyl group thereby
obtaining a racemic mixture of diketides coupled to said
benzoxazolone.
30. The tailored polyketides of claim 16 selected from the group
consisting of those of Examples 17B and 17C.
31. The derivatized polyketide or tailored polyketide of claim 19
which is selected from the group consisting of the polyketides of
Examples 16A, 16B, 18 and 24.
Description
[0001] This application claims priority under 35 U.S.C. .sctn.119
from U.S. Ser. No. 60/117,384 filed Jan. 27, 1999. The contents of
this application are incorporated herein by reference.
TECHNICAL FIELD
[0002] The invention concerns methods for the efficient synthesis
of oligoketide thioesters, including diketide and triketides, which
are useful as intermediates in polyketide production and to methods
to use these intermediates. The methods of synthesis are suitable
for liquid phase as well as solid-phase combinatorial synthesis.
The invention also includes polyketide and tailored polyketide
products.
BACKGROUND OF THE INVENTION
[0003] The creation of novel macrolide polyketides has been
achieved through genetic manipulation of polyketide synthases. The
modular nature of the Type 1 polyketide synthases allows for domain
exchange between different polyketide synthase genes, resulting in
hybrid genes which produce polyketide synthases with altered
properties that result, in turn, in modified macrolide structures.
Thus, it is possible to control chain length, choice of chain
extender unit, degree of .beta.-carbon oxidation level, and to some
degree stereochemistry. The choice of starter unit has been more
difficult to control. Two complementary approaches have been
described.
[0004] Dutton, et al., J. Antibiotics (1991) 44:357-365
demonstrated that the avermectin polyketide synthase was somewhat
flexible in choice of starter units. When denied the natural
starter unit through inactivation of the branched-chain amino acid
dehydrogenase, the avermectin polyketide synthase will accept a
variety of .alpha.-branched carboxylic acids as the starter unit.
However, only about 30 acids out of nearly 800 candidate acids
tried were accepted. Acids without an .alpha.-branch appear to be
metabolized through .beta.-oxidation until an .alpha.-branch is
reached, further limiting this methodology. Marsden, et al.,
Science (1998) 79:199-202 exchanged the native loading domain of
the erythromycin PKS with that from the avermectin polyketide
synthase, resulting in a hybrid PKS having the same loosened
starter unit specificity as the avermectin PKS. Clearly, the native
specificities of enzymatic domains will always be a limitation on
the flexibility of resulting hybrid systems.
[0005] A more general method for controlling starter unit
specificity has been described by Jacobsen, et al., Science (1997)
277:367-369. Inactivation of the ketosynthase in module 1 (KS1) of
the erythromycin PKS (DEBS) results in an enzyme (KS1.degree.-DEBS)
incapable of initiating polyketide synthesis using precursors
normally available to the cell. When supplied with a suitable
thioester of the diketide product of module 1 or its analogs,
however, KS1.degree.-DEBS efficiently incorporates these into
full-length polyketides. Subsequent experiments have demonstrated
that a very wide range of diketide analogs are accepted by
KS1.degree.-DEBS, making this a very general method for production
of analogs of the polyketide precursor of erythromycin,
6-deoxyerythronolide B (6-dEB), with variations at the positions
controlled by the starter unit. Further, this method allows for
production of 6-dEB analogs altered at the 12-position; this is
equivalent to altering the substrate specificity of the module 1
acyltransferase (AT1) which transfers the first extender unit.
While this has been accomplished through the above described domain
exchange experiments as well, the "diketide method" allows for
introduction of 12-position substituents which are not available
from nature. Furthermore, triketide analogs are accepted, opening
the 10- and 11-positions of 6-dEB for modification. The 6-dEB
analogs obtained can be further converted into analogs of
erythromycin by feeding to a suitable converter strain, such as a
strain of Saccharopolyspora erythraea containing a non-functional
erythromycin PKS. The resulting erythromycins have altered
side-chains at the 13-position as well as other optional
modifications, and show altered biological activity. These
erythromycin analogs can also be produced by introducing the
KS1.degree.-mutation into an erythromycin-producing strain of
Saccharopolyspora erythraea, then supplying the mutant strain with
suitable diketide or triketide thioesters as described above.
[0006] Implementation of this method requires the availability of
the N-acylcysteamine oligoketide thioesters. Synthetic methods
available in the art for these thioesters do not lend themselves to
efficient, economical synthesis, or to the systematic production of
variants. The diketide and triketides also typically contain chiral
centers requiring the control of absolute or relative
stereochemistry.
[0007] Cane, D. E., et al., J Am Chem Soc (1987) 109:1255-1257
describes a three-step process to produce the N-acetylcysteamine
thioester of (2S, 3R)-2-methyl-3-hydroxy pentanoic acid. The method
relies on the use of a chiral reagent
N-propionyl-(4S)-4-isopropyl-2-oxazolidinone for control of the
absolute stereochemistry of the product: 1
[0008] N-propionyl-(4S)-4-isopropyl-2-oxazolidinone
[0009] This material results from the acylation of
(4S)-4-isopropyl-2-oxaz- olidinone with propionyl chloride,
typically using a strong base such as n-butyllithium at low
temperature. The Cane process is an aldol condensation of this
starting material with propionaldehyde in the presence of
dibutylboron triflate (Bu.sub.2BOTf), followed by hydrolysis of the
imide (lithium hydroperoxide) and thioesterification with
N-acetylcysteamine in the presence of diphenyl phosphorylazide and
triethylamine. This multi-step process is inefficient, with
substantial losses accompanying the hydrolysis step.
[0010] Cane, D. E., et al., J Antibiotics (1995) 48:647-651 was
able to improve yields using a five-step process which replaces the
aldol condensation with a Claisen condensation between the lithium
enolate of the propionyl oxazolidinone
(N-propionyl-(4S)-4-benzyl-2-oxazolidinone was used as the
stereochemistry controlling starting material in this method) and
propionyl chloride followed by reduction of the resulting
.beta.-ketoester product using zinc borohydride. Protection of the
.beta.-hydroxy substituent as a tert-butyldimethylsilyl ether
preceded hydrolysis of the imide, which again required lithium
hydroperoxide. The protecting group gave improved yields from
hydrolysis, but required an additional two steps to add and remove.
This longer process also suffers from the use of zinc borohydride,
which is not commercially available.
[0011] Cleavage of the N-acyloxazolidinones resulting from either
aldol or Claisen condensations as described above is problematic.
Various methods of cleavage are known in the art, including that of
Evans, D. A., et al., Tetrahedron Lett (1987) 28:6141, in which
undesired reaction at the oxazolidinone carbonyl during hydrolysis
is suppressed by the use of lithium hydroperoxide. This process
requires the use of concentrated solutions of hydrogen peroxide,
which are explosive and dangerous for large-scale processes. The
N-acyloxazolidinones are unreactive towards thiols or thiolates,
although some conversion to thioesters can be observed using
concentrated solutions of lithium thiolates in tetrahydrofuran. The
low solubility of the thiolates in tetrahydrofuran combined with
epimerization of chiral diketides due to the basicity of the
thiolates limits the utility of this method. Miyata, O., et al.,
Syn Lett (1994) 637-638, describes conversion of
N-acyloxazolidinones to S-benzylthioesters through the use of
lithium benzylthiotrimethylaluminat- e. The production of more
complex thioesters containing groups capable of binding Lewis acids
like trimethylaluminum, such as those based on N-acylcysteamine,
has not been reported.
[0012] The N-acetylcysteamine thioesters of larger oligoketides
have also been prepared. Cane, D. E., et al., J Am Chem Soc (1993)
115:522-526 synthesized the N-acetylcysteamine thioester of (4S,
5R)-5-hydroxy-4-methyl-2-heptenoic acid using the stereochemically
controlled aldol condensation product of
N-propionyl-(4S)-4-benzyl-2-oxaz- olidinone as the starting
material: 2
[0013] This imide was converted to the corresponding aldehyde, and
extended at the carbonyl group by a Wittig reaction to obtain the
desired triketide as the ethyl ester which was then hydrolyzed and
converted to the acylcysteamine thioester in a two-step process.
Yields were improved by addition of steps protecting the alcohol,
Cane, D. E., et al., J Am Chem Soc (1993) 115:527-535. However,
this approach clearly does not lend itself to efficient modular
solid-phase synthesis since the building of the triketide chain is
nonlinear--i.e., the condensations and the Wittig reactions extend
the diketide in opposing directions. Each new analog requires
complete passage through the synthesis with no common
intermediates.
[0014] While it is clear that thioester forms of acyl moieties,
diketides and triketides can be incorporated by PKS systems, to
date, little has been reported concerning the optimal thioesters to
produce the desired polyketides other than that N-acetylcysteamine
thioesters are generally effective as compared with the free
carboxylic acids or their oxy-esters (Cane & Yan). It may be
expected, however, that the nature of the thioester, e.g. the acyl
group in an N-acylcysteamine thioester, might influence such
important factors as water solubility, transport into the bacterial
cell, metabolism, and recognition by the PKS. A synthetic method
for producing variation in the thioester group itself would thus be
advantageous.
[0015] The present invention offers both improved efficiency in the
synthesis of optically-pure diketide thioester intermediates and an
approach which provides for efficient extension of the diketides
into the corresponding triketide thioesters and provides for
additional condensation steps to extend the oligoketide still
further. The present invention further provides a method for
synthesis of racemic rather than optically pure diketide
thioesters. The racemic materials constitute low-cost alternatives
for large-scale production of novel polyketides by
fermentation.
DISCLOSURE OF THE INVENTION
[0016] The invention offers improvements in the synthesis of
oligoketide thioester intermediates. These intermediates can then
be incorporated into pathways for the synthesis of novel
polyketides using native or modified polyketide synthase (PKS)
systems. The invention offers an improvement in the efficiency of
diketide synthesis as well as a method for synthesis of triketides
and oligoketides in general which is adapted to efficient, linear,
solid-phase synthesis. The invention further provides a method to
produce racemic diketide thioesters in an economical manner. The
invention further provides a method for producing novel polyketides
suitable for further modification through the introduction of
unique functionalities.
[0017] Thus, in one aspect, the invention is directed to the
conversion of an acyl imide such as that of a diketide, triketide
or oligoketide directly to an N-acylcysteamine thioester by
treating the imide with a salt of the corresponding mercaptan. For
the synthesis of optically active oligoketide thioesters starting
from chiral oxazolidinones, this is done in the presence of a Lewis
acid to facilitate the reaction and preserve stereochemical purity.
For the synthesis of racemic oligoketide thioesters starting from
achiral benzoxazolones, the Lewis acid is not required. This method
obviates the intermediate steps of imide hydrolysis, alcohol
protection, thioesterification, and deprotection used by previous
methods. This method is particularly advantageous for solid-phase
synthesis, as it allows for generation of the product with
simultaneous cleavage of the oligoketide from the solid support. A
particularly facile process using transthiolation of thioesters is
given.
[0018] In a second aspect, the invention is directed to a method to
synthesize racemic diketides and their derivatives through the
titanium-mediated aldol condensation between
N-acyl-2-benzoxazolones with aldehydes, followed by reaction of the
aldol products with nucleophiles to yield the desired derivatives.
This method provides a direct route to various oligoketide
derivatives, including esters and amides, and is particularly
advantageous for the multi-kilogram, economical synthesis of
diketide N-acylcysteamine thioesters required for fermentation. As
the relative chirality of the carbons at positions 2 and 3 of the
attached acyl group is preserved, the racemic mixture will contain
one isomer which can be utilized by the PKS and only one additional
isomer which cannot. This is in contrast to production of the four
possible diestereomers which would result in utilization of only
one-quarter of the available molecules.
[0019] Thus, in still another aspect, the invention is directed to
methods to synthesize diketides and triketides which can be used to
produce macrolides with functional substituents for example at the
13- and 14-positions by employing, for example, alkenyl- or
benzyloxy-aldehydes to introduce starter unit and/or first extender
moiety equivalents containing derivatizable groups. The benzyloxy
group can readily be converted to a hydroxyl by reduction and then
mesylated to provide a suitable leaving group for replacement with
nucleophiles, including halides, azides, amines, thiols, other
alcohols, and cyanide. The alkenyl group can be functionalized by
any of numerous methods known in the art, including Heck coupling
to introduce aryl groups. Such derivatizations can be performed
either on the oligoketide or on the polyketides which are produced
upon feeding of the oligoketides to suitable PKS systems or
cultures of microorganisms.
[0020] In an additional aspect, the invention is directed to
methods to synthesize oligoketide thioesters using solid-phase
combinatorial chemistry. These methods are particularly
advantageous when a library of oligoketide thioesters is
desired.
[0021] In summary, because the invention permits a wide variety of
diketide and triketide thioesters to be synthesized in a facile and
economic manner, it is possible to prepare a wide variety of
polyketides and their tailored derivatives taking advantage of the
availability of both recombinant and natively produced polyketide
synthase systems and tailoring enzymes, as well as employing
chemical transformations using side-chain functional groups.
[0022] In still other aspects, the invention relates to feeding
diketides or triketides, prepared by the methods of the invention,
to suitable PKS systems in vitro or in vivo to obtain oligoketides
or polyketides and further converting said polyketides to
antibiotics by glycosylation and/or other modifications. The
invention also relates to novel intermediates and the resulting
modified polyketides and antibiotics.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 sets forth structures of illustrative suitable N-acyl
cysteamines.
[0024] FIG. 2 illustrates the method for conversion of
N-acyloxazolidinones into N-acylcysteamine thioesters.
[0025] FIG. 3 illustrates methods for the conversion of
N-acyl-2-benzoxazolones into various acyl derivatives, including
N-acylcysteamine thioesters.
[0026] FIG. 4 illustrates the transthioesterification method
developed for use with the diketide benzoxazolones.
[0027] FIG. 5 illustrates the formation of N-acyl-2-benzoxazolone
and the aldol condensation between N-acyl-2-benzoxazolones and
aldehydes, used to prepare intermediates for the synthesis of
racemic diketides.
[0028] FIG. 6 illustrates the rationale for enforcement of
syn-stereochemistry by the benzoxazolone auxiliary.
[0029] FIGS. 7A and 7B illustrate typical diketides, shown as their
N-acylcysteamine (SNAC) thioesters, prepared according to the
invention.
[0030] FIG. 8 illustrates synthesis of oligoketide thioesters using
solid-phase chemistry.
MODES OF CARRYING OUT THE INVENTION
[0031] The invention provides methods useful in the synthesis of
intermediates for the production of polyketides that have
characteristics desirable for efficient and practical
applications.
[0032] An efficient synthetic strategy for the required oligoketide
thioesters should provide:
[0033] 1) stereochemical control;
[0034] 2) a minimum number of synthetic steps;
[0035] 3) synthetic steps with high yields;
[0036] 4) use of common intermediates;
[0037] 5) adaptability to solid phase synthesis;
[0038] 6) adaptability to combinatorial library generation.
[0039] For large-scale applications, further criteria concern the
cost and availability of reagents.
[0040] The invention methods provide these characteristics. The
crucial step in all routes to diketide thioesters is the formation
of the thioester linkage. Due to low reactivity of the commonly
used N-acyloxazolidinone intermediates towards thiol nucleophiles,
this process usually requires several steps as described above. The
oxazolidinone auxiliary is removed by hydrolysis, and the resulting
acid is activated and converted into the thioester. It is possible
to convert N-acyloxazolidinones into thioesters directly by
treatment with the lithium salt of the mercaptan, but yields are
typically low and loss of stereochemical integrity is often noted
for chiral diketides. The invention provides an efficient method
for direct conversion of N-acyloxazolidinones into thioesters of
N-acylcysteamines which uses the trimethylaluminum complex of the
lithium mercaptide (FIG. 2). This proceeds in high yields without
detectable loss of stereochemical integrity. Since the filing of
provisional application 60/117,384, a similar method using
trimethylaluminum with N-acetylcysteamine has been reported in C.
LeSann, et al., Tetrahedron Letters (1999) 40:4093-4096.
[0041] The invention also provides a method for the direct
conversion of N-acyl-2-benzoxazolones into N-acylcysteamine
thioesters by simple treatment with an alkali metal salt of the
mercaptan in an alcohol solvent (FIG. 3). This is efficient and
mild due to the more ready displaceability of the benzoxazolone as
compared with an oxazolidinone and the lower basicity of thiolates
in protic solvents. Methods for the conversion of the
N-acylbenzoxazolones into other functional groups are also
illustrated. The alkali metal salt of the mercaptan may be
generated through the reaction of a mercaptan with a metal
alkoxide, such as sodium methoxide or sodium ethoxide, under inert
atmosphere so as to prevent disulfide formation. In a particularly
simple embodiment, the required alkali metal salt of the mercaptan
is generated in situ through treatment of a alcoholic solution of a
simple thioester, e.g., N,S-diacylcysteamine, with one molar
equivalent of an alkali metal alkoxide. Addition of the
N-acylbenzoxazolone where acyl is an oligoketide then provides the
oligoketide thioester. Suitable alcohols are methanol, ethanol,
isopropanol, and related solvents. Suitable alkali metal alkoxides
are those derived from the aforementioned alcohol solvents, such as
lithium methoxide, sodium methoxide, potassium methoxide, and
similar salts of the other alcohols. The reaction is typically
performed at ambient temperatures. This method has the advantage of
avoiding disulfides which are typically present in free mercaptans
due to air oxidation. The N,S-diacylcysteamines are readily
available through the reaction of cysteamine hydrochloride with an
excess of the acyl anhydride in water in the presence of a suitable
base. A convenient base is saturated aqueous sodium bicarbonate,
which provides a pH where the thioester product is stable. Unlike
N-acylcysteamines, the N,S-diacylcysteamines are typically
crystalline, stable materials which can be stored indefinitely.
[0042] The invention further provides a method for synthesis of
racemic diketides using 2-benzoxazolone as supporting auxiliary
(FIG. 4). The titanium tetrachloride-mediated aldol condensation
between N-propionyl-2-benzoxazolone and an aldehyde provides high
yields of these diketides, with excellent diastereochemical
control. "Benzoxazolone derivative" means, generally, the imide of
an acyl group with a 2-benzoxazolone. The aromatic moiety in
benzoxazolone may be unsubstituted as in benzoxazolone per se or
may be substituted, for example as is the case for chlorzoxazone.
Alternative substitutions on the benzylidene moiety may also be
employed, such as bromo, methyl, and the like. Both 2-benzoxazolone
and 5-chloro-2-benzoxazolone (chlorzoxazone) have been shown to be
effective auxiliaries, supporting >95% syn aldol condensation
for simple aldehydes and ca. 90% syn aldol condensation with
sterically-hindered aldehydes such as pivalaldehyde and with
chelating aldehydes such as .alpha.-alkoxyaldehydes. The titanium
aldol condensation has further advantages in that it can be
performed at moderate temperatures (0.degree. C.), unlike reactions
of lithium enolates which require the use of -78.degree. C., and in
that the reagents are extremely inexpensive ($10 /mol) as compared
with dibutylboron triflate ($750 /mol). Further, the oxidative
workup using concentrated hydrogen peroxide needed with
boron-mediated aldols is not required.
[0043] The use of N-propionyl-2-benzoxazolone in the aldol
condensation provides diketides of benzoxazolones having a 2-methyl
substituent, which, in turn, provides a 12-methyl group in the
6-dEB analog obtained upon conversion of the diketide by the
erythromycin PKS. Similarly, the use of N-crotonyl-2-benzoxazolone
ultimately provides diketides having a 2-vinyl substituent, which
provides a 12-vinyl group in the 6-dEB analog obtained upon
conversion of the diketide by the erythromycin PKS. Other
N-acyl-2-benzoxazolones can be used to provide other 2-substituted
diketides, and thus other 12-substituted 6-dEB and erythromycin
analogs.
[0044] The invention further provides a method for introducing
substituents at the 12-, 14-, and 15-positions of 6-dEB or
erythromycin which are not tolerated by the erythromycin PKS and
thus cannot be introduced directly by feeding the corresponding
oligoketide thioester. This method involves feeding an oligoketide
thioester containing a functional group, typically an alkene or a
protected alcohol group, which is tolerated by the PKS and which
can be converted post-PKS into the desired functionality using
chemical, enzymic, or biological conversion. For example, the
erythromycin PKS will efficiently convert diketides containing
alkene groups either at the 2- or 3-positions (or both) to provide
the corresponding 12- or 13-vinyl 6-dEB analogs. The erythromycin
PKS will convert 3-hydroxy-2-methyl-4-pentenoate N-acylcysteamine
thioesters into 14,15-dehydro-6-dEB, for example, and the post-PKS
enzymes of Saccharopolyspora erythraea will convert this further
into 14,15-dehydroerythromycins. This introduces a unique alkene
functionality into the 6-dEB and erythromycin molecules. Methods
for conversion of this alkene, e.g., into halides, carbonyls,
alcohols, ethers, and amines are well known in the art. The alkene
can also be used to add aromatic moieties onto the 6-dEB or
erythromycin molecule through the Heck reaction: 3
[0045] Similarly, the erythromycin PKS will convert
3-hydroxy-2-vinylpentanoate N-acylcysteamine thioesters into
12-desmethyl-12-vinyl-6-dEB. This provides a unique alkene
functionality at the 12-position of 6-dEB which can be further
manipulated. As an extension of this concept, Streptomyces
coelicolor CH999 expressing the plasmid pJRJ2 converts
3-hydroxy-2-vinyl-6-heptenoate N-acylcysteamine thioesters into
12,15-divinyl-12-desmethyl-6-dEB at levels of approximately 50
mg/L: 4
[0046] This compound can be subsequently converted into various
derivatives, such as the bicyclic analog illustrated through the
use of an olefin metathesis catalyst.
[0047] Other protected or masked functionalities can be introduced
into the 6-dEB and erythromycin molecules in this fashion. For
example, alcohols protected as esters or as benzyl ethers would be
suitable precursors which would allow for introduction of a new
alcohol group in the polyketide. The modification of alcohols into
other functional groups is well known in the art.
[0048] This methodology can also be used to introduce reactive
functionalities directly. As an example, the erythromycin PKS will
convert 5-halo-3-hydroxy-2-methylpentanoate N-acylcysteamine
thioesters into the corresponding 15-halo-6-dEBs. The halogen can
be F, Cl, Br, or I, and supplies a readily-displaceable group for
subsequent modification of the 15-position of the 6-dEB or
erythromycin.
[0049] It can be seen, therefore, that the feeding of synthetic
diketide thioester analogs to the erythromycin PKS or an organism
expressing the erythromycin PKS is a useful means of producing
novel polyketides. The method is particularly useful when the PKS
has been modified so as to preclude formation of the natural
polyketide, such as by inactivation of the module 1 ketosynthase,
or more generally when the supply of natural starter unit has been
otherwise eliminated.
[0050] It can be seen as well that methods existing in the art for
construction of lengthier oligoketides can be adapted for use in
these systems. For example, triketides are readily available by
aldol condensations between aldehydes and .beta.-ketoimides as
described in D. A. Evans, et al., J. Am. Chem. Soc. (1990)
112:866-868. Methods have been developed for the efficient control
of relative stereochemistry in these transformations, as described
in D. A. Evans, et al., Tetrahedron (1992) 48:2127-2142. The
stereoselective reduction of the resulting products using
triacetoxyborohydrides has been described in D. A. Evans, et al.,
J. Am. Chem. Soc. (1988) 110:3560-3578, and provides a means of
further altering the functionality of the oligoketides by selective
introduction of a .beta.-hydroxyl. Such hydroxyls can be further
converted into alkenes through acylation and .beta.-elimination,
with the proviso that other hydroxyls in the oligoketide must be
protected against acylation or at least must be readily deprotected
afterwards. The invention provides a particularly simple method for
this transformation using phosgene to form a cyclic carbonate,
which simultaneously activates the .beta.-hydroxyl for elimination
and protects the .delta.-hydroxyl:
[0051] Construction of triketides: 5
[0052] Alternatively, the alkene resulting from elimination can be
reduced to the alkane, for example by catalytic hydrogenation,
prior to thiolysis. Thus, all four reductive outcomes observed from
natural polyketide synthesis can be mimicked in the chemical
construction of triketides.
[0053] The direct, efficient conversion of oligoketide imides into
thioesters opens the possibility of efficient solid-phase synthesis
of oligoketide thioesters, as thiolysis can be used to free the
oligoketide chain from the solid support as the final step in the
synthesis. Methods exist for the linear elaboration of oligoketides
wherein the oligoketide chain is grown off an initial auxiliary
unit, typically a chiral oxazolidinone, at least up to the
triketide level. The number of required steps is minimal and the
yields are high. The use of the 4-benzyl-2-oxazolidinone residue
maintains the stereochemistry through multiple chain extensions
using similar reagents. Because common intermediates are used and
the auxiliary stereochemistry controlling compound can readily be
linked to solid supports as described in the invention, the method
provides a suitable basis for the solid-phase production of
combinatorial libraries of triketides and beyond.
[0054] Two possible attachment sites to a solid support can be
envisioned. By providing functionality on the phenyl group of the
4-benzyloxazolidinone, covalent coupling to a wide variety of
supports may conveniently be obtained; means for coupling through
this moiety are well known in the art. For example, the
corresponding auxiliary derived from tyrosine rather than
phenylalanine can be readily prepared. This would provide a
phenolic hydroxyl group which could readily be attached to a solid
support through, for example, reaction with a chlorobenzyl
polystyrene resin to give a diphenylether-linked chiral
oxazolidinone. 6
[0055] The oligoketide chain can be grown on this support using
methods well-established in the art, then cleaved from the solid
support, preferably by formation of the thioester as described
herein. This offers an advantage over previous methods for
solid-phase oligoketide synthesis, e.g., Reggelin, M., et al.,
Tetrahedron Letters (1996) 37:6851-6852, in which the oligoketide
chain itself is used as the attachment point, with a corresponding
attachment functionality remaining as part of the oligoketide at
the end of the synthesis.
[0056] Alternatively, the oxazolidinone ring itself is used as the
point of attachment to the support. For example, the solid support
of the invention may employ a chiral imidazolidinone, wherein the
imidazolidinone replaces the oxazolidinone described above: 7
[0057] The second nitrogen atom of the imidazolidinone is used as
the attachment point to the solid support, leaving the nitrogen
adjacent to the chiral center (equivalent to the nitrogen in an
oxazolidinone) open for acylation with an acyl chloride. The use of
untethered chiral imidazolidinones as synthetic auxiliaries has
been described by S. E. Drewes, et al., Chem. Ber. (1993) 126:2663,
and an especially facile method for their acylation has been
described by W. M. Clark & C. Bender, J. Org. Chem. (1998)
63:6732.
[0058] The tethered chiral imidazolidinones can be readily prepared
from optically pure amino acids by standard procedures; e.g., by
conversion of a chiral amino acid such as phenylalanine into the
carbamate by reaction with a suitable chloroformate or
chloroformate equivalent, followed by conversion to the
aminoaldehyde (Rittel, K. E., et al., J Org Chem (1988) 47:3016;
Organic Syntheses, vol. 67:69-75) and subsequent reductive
amination to add a suitable functionalized linker. 8
[0059] The functional group, shown as "X" is, for example, an
amine, a carboxylate or an ester, thiol or halide which is used to
attach the auxiliary to a solid support. The resulting amino
carbamate is cyclized to the imidazolidinone by treatment with a
suitable base or with heat.
[0060] Racemic diketides can be synthesized on solid supports using
a similar technique. In this case, the 2-benzoxazolone auxiliary
can be attached to the support by either of two methods.
Halogenated benzoxazolones, such as chlorzoxazone
(5-chloro-2-benzoxazolone), are readily available and provide a
simple means of attachment through the aromatic halide. For
example, chlorzoxazone can be coupled with an alkene-containing
support using palladium catalysis (the Heck reaction). Alternately,
2-benzimidazolone (2-hydroxybenzimidazole) can be coupled to a
support through one of the imidazolone nitrogens, leaving the
second free for acylation as described above. 9
[0061] The methods described above for elaboration of triketides
are ideally suited to solid-phase synthesis, as the directing
auxiliary group (oxazolidinone or benzoxazolone) remain attached to
the growing oligoketide chain. The attached auxiliary then serves
as a leaving group during thioester formation, yielding an
oligoketide thioester with no residue remaining from the solid
support.
[0062] Incorporation into Polyketides
[0063] As used herein, "polyketide" refers to the immediate product
of a polyketide synthase enzyme system. It is generally a lactone
of 13-15C. An example of a polyketide would be 6-dEB, the immediate
product of the erythromycin PKS. "Tailored polyketides" refers to
the products of subsequent derivatization of the resultant
polyketide which occurs through enzymatic treatment by enzymes
endogenous to organisms which synthesize polyketide antibiotics.
Such tailoring enzymes may add hydroxyl groups, remove hydroxyl or
oxo groups, add sugars, modify sugars that have been coupled to the
polyketide, and the like. "Derivatized polyketides" refers to
polyketides or tailored polyketides which have been modified
chemically in ways generally unavailable from straightforward
enzymatic treatment. The diketides and triketides prepared by the
methods of the invention, because they contain functional groups
which can further be reacted result in polyketides and tailored
polyketides that can be derivatized using synthetic chemical
reactions. Methods for further converting polyketides (or tailored
polyketides) are found, for example, in PCT publications WO
99/35156 and WO 99/35157, incorporated herein by reference. Such
methods are also described in U.S. Ser. Nos., respectively,
60/172,154 and 60/172,159, both filed Dec. 17, 1999; 60/173,805
filed Dec. 30, 1999; and 60/173,804 filed Dec. 30, 1999, and each
incorporated herein by reference.
[0064] The thioesters of the diketides and triketides of the
invention can be incorporated into polyketides by the PKS system,
most advantageously when competition from the native starter unit
is eliminated by, for example, the inactivation of the ketosynthase
domain in module 1 as described in PCT application PCT/US96/11317
incorporated herein by reference. Polyketide synthases thus
modified are also described in U.S. Ser. No. 08/896,323 filed July
17, 1997 and incorporated herein by reference. As described in
these applications, the polyketide synthase system can be employed
in a cell-free context, or can be utilized in vivo either in its
native host or in a recombinant host cell. For example, the
organism which natively produces erythromycin, Saccharopolyspora
erythreae may be used, or, as set forth in U.S. Pat. No. 5,672,491,
the entire erythromycin gene cluster can be inserted into a
suitable host such as Streptomyces coelicolor or S. lividans,
preferably a S. coelicolor or S. lividans which has been modified
to delete its endogenous actinorhodin polyketide synthesis
mechanism. A typical host would be S. coelicolor CH999/pJRJ2, which
expresses a mutant 6-deoxyerythronolide B synthase having an
inactivated module 1 ketosynthase (J. Jacobsen, et al., 1997
Science 277:367-369). The diketides or triketides are thus
incorporated into the resulting polyketide. In the case of the
diketides and triketides provided by this invention, the resulting
erythronolide will be correspondingly modified at positions 10-15.
For example, feeding a growing culture of S. coelicolor CH999/pJRJ2
with (2S,3R)-5-fluoro-3-hydroxy-2-methylpentanoate
N-acetylcysteamine thioester results in production of
15-fluoro-6-deoxyerythronolide B, while feeding with
(2S,3R)-3-hydroxy-2-methylhexanoate N-acetylcysteamine thioester
results in production of 15-methyl-6-deoxyerythronolide B. Feeding
S. coelicolor CH999/pJRJ2 with (2S,3R)-3-hydroxy-2-vinylpentanoate
N-acetylcysteamine thioester results in production of
12-desmethyl-1 2-vinyl-6-deoxyerythron- olide B.
[0065] Further, the diketide or triketide intermediates can be
provided to PKS enzymes other than the 6-dEB synthase of
Saccharopolyspora erythraea. Other PKS enzymes include the 6-dEB
synthase of Micromonospora megalomicea and its KS1.degree.
derivative described in U.S.S.N. 60/158,305, filed Oct. 8, 1999;
the oleandolide PKS and its KS1.degree. derivative described in PCT
application No. US99/24478, filed Oct. 22, 1999; and the
narbonolide PKS and its KS1.degree. derivative described in PCT
publication No. WO 99/61599, published Dec. 2, 1999, all
incorporated by reference.
[0066] The diketides and triketides can be provided to a host cell
that expresses a PKS but not post PKS modification enzymes (such as
hydroxylases and glycosyltransferases) or can be provided to a host
cell that expresses both types of enzymes.
[0067] Recombinant host cells containing cloned PKS expression
vectors can be constructed to express all of the biosynthetic genes
for a modified polyketide or only a subset of the same. If only the
genes for the PKS are expressed in a host cell that otherwise does
not produce polyketide modifying enzymes that can act on the
polyketide produced, then the host cell produces unmodified
polyketides. Such unmodified polyketides can be hydroxylated and
glycosylated, for example, by adding them to the fermentation of a
strain such as, for example, Streptornyces antibioticus or
Saccharopolyspora erythraea, that contains the requisite
modification enzymes.
[0068] If desired, further modifications at positions 14 and 15 are
achievable once the resulting polyketide is isolated by employing
an appropriate benzyloxy-, alkene-, or halo-substituted diketide
thioester. As set forth above, these groups can be converted to
other functionalities using methods well known in the art.
[0069] The resulting polyketides can further be modified by
chemical means or by feeding to a native antibiotic producing host
for glycosylation or further modification. For example, a resulting
6-deoxyerythronolide can be fed to Sac. erythraea for hydroxylation
at the 6- and/or 12-positions and sugar attachment at the 3- and/or
5-positions. This is particularly useful when the organism used
contains a defective PKS gene, resulting either from random
mutagenesis or from designed deletion. The strain Sac. erythraea
K39-14 expresses a defective 6-deoxyerythronolide B synthase, and
so is incapable of producing erythromycins under normal
fermentation conditions. Feeding a growing culture of Sac.
erythraea K39-14 with 15-fluoro-6-deoxyerythonolide B results in
production of 15-fluoroerythromycins. Feeding this strain with
15-methyl-6-deoxyerythro- nolide B results in formation of
15-methylerythromycins. Both 15-fluoroerythromycin A and
15-methylerythromycin A have been found to have strong
antibacterial activity.
[0070] In lieu of, or in addition to chemical synthesis steps, the
initially produced polyketides can be "tailored." There is a wide
variety of diverse organisms that can modify polyketides and/or
their derivatives to provide compounds with, or that can be readily
modified to have, useful activities. As stated above,
Saccharopolyspora erythraea can convert 6-dEB to a variety of
useful compounds. The erythronolide 6-dEB is converted by the eryF
gene product to erythronolide B, which is, in turn, glycosylated by
the eryB gene product to obtain 3-O-mycarosylerythronolide B, which
contains L-mycarose at C-3. The enzyme eryC gene product then
converts this compound to erythromycin D by glycosylation with
D-desosamine at C-5. Erythromycin D, therefore, differs from 6-dEB
through glycosylation and by the addition of a hydroxyl group at
C-6. Erythromycin D can be converted to erythromycin B in a
reaction catalyzed by the eryG gene product by methylating the
L-mycarose residue at C-3. Erythromycin D is converted to
erythromycin C by the addition of a hydroxyl group at C-12 in a
reaction catalyzed by the eryK gene product. Erythromycin A is
obtained from erythromycin C by methylation of the mycarose residue
in a reaction catalyzed by the eryG gene product. The unmodified
polyketide compounds provided by the present invention can be
provided to cultures of S. erythraea and converted to the
corresponding derivatives of erythromycins A, B, C, and D in
accordance with the invention. To ensure that only the desired
compound is produced, one can use an S. erythraea eryA mutant that
is unable to produce 6-dEB but can still carry out the desired
conversions (Weber, et al., 1985, J. Bacteriol. 164(1):425-433).
Also, one can employ other mutant strains, such as eryB, eryC,
eryG, and/or eryK mutants, or mutant strains having mutations in
multiple genes, to accumulate a preferred compound. The conversion
can also be carried out in large fermentors for commercial
production.
[0071] There are other useful organisms that can be employed to
hydroxylate and/or glycosylate the compounds of the invention. The
organisms can be mutants unable to produce the polyketide normally
produced in that organism, the fermentation can be carried out on
plates or in large fermentors, and the compounds produced can be
chemically altered after fermentation. Thus, Streptomyces
venezuelae, which produces picromycin, contains enzymes that can
transfer a desosaminyl group to the C-5 hydroxyl and a hydroxyl
group to the C-12 position. In addition, S. venezuelae contains a
glucosylation acticity that glucosylates the 2'-hydroxyl group of
the desosamine sugar. This latter modification reduces antibiotic
activity, but the glucosyl residue is removed by enzymatic action
prior to release of the polyketide from the cell. Another organism,
S. narbonensis, contains the same modification enzymes as S.
venezuelae, except the C-12 hydroxylase. Thus, the present
invention includes the compounds produced by hydroxylation and
glycosylation of the initially formed polyketides of the invention
by action of the enzymes endogenous to S. narbonensis and S.
venezuelae.
[0072] Other organisms suitable for making compounds of the
invention include Micromonospora megalomicea, Streptomyces
antibioticus, S. fradiae, and S. thermotolerans. S. antibioticus
produces oleandomycin and contains enzymes that hydroxylate the C-6
and C-12 positions, glycosylate the C-3 hydroxyl with oleandrose
and the C-5 hydroxyl with desosamine, and form an epoxide at
C-8-C-8a. S. fradiae contains enzymes that glycosylate the C-5
hydroxyl with mycaminose and then the 4'-hydroxyl of mycaminose
with mycarose, forming a disaccharide. S. thermotolerans contains
the same activities as S. fradiae, as well as acylation activities.
Thus, the present invention provides the compounds produced by
hydroxylation and glycosylation of the macrolide aglycones of the
invention by action of the enzymes endogenous to S. antibioticus,
S. fradiae and S. thermotolerans. The modified polyketides of the
invention can also be produced in recombinant host cells that have
been transformed with genes that encode polyketide modification
enzymes from another organism.
[0073] The present invention also provides methods and genetic
constructs for producing the glycosylated and/or hydroxylated
compounds of the invention directly in the host cell of interest.
Thus, the polyketides of the invention can be produced directly by
feeding in Saccharopolyspora erythraea, Streptomyces antibioticus,
Micromonospora megalomicea, S. fradiae, and S. thermotolerans. A
number of erythromycin high-producing strains of Saccharopolyspora
erythraea have been developed, and such strains can also be used to
feed the diketide compounds of the invention to produce modified
polyketides.
[0074] Modification can also be effected by chemical means, such as
glycosylation through cell-free preparations of appropriate
glycosylases or through chemical derivatization. Thus, a
multiplicity of polyketides and corresponding antibiotics may be
obtained using the methods and compounds of the invention.
[0075] In a specific embodiment of the invention, the diketide
thioester prepared from 4-pentenal is used to produce
15-ethenylerythromycins, which can be chemically converted into
15-(2-arylethyl)erythromycin analogs such as
15-(2-(3-quinolyl)ethyl)erythromycin A and related compounds:
10
[0076] These analogs are expected to provide an aromatic moiety
suitably positioned to interact with additional binding sites on
the bacterial ribosome, and thus exhibit enhanced antibacterial
activity. Particularly preferred analogs are the
6-O-methyl-3-descladinosyl-3-oxo analog and the corresponding
11,12-cyclic carbamate (X.dbd.H,F): 11
[0077] Given the high structural similarity between the modular
polyketide synthases examined to date, it should be clear that the
invention will provide methods for production of novel polyketides
using many different enzymes other than the erythromycin polyketide
synthase. For example, the genes encoding the polyketide synthases
for rapamycin, FK-506, soraphen, epothilone, rifamycin, picromycin,
tylosin, spiramicin, niddamycin, and avermectin have been examined
and found to show high homologies.
[0078] The following examples are thus intended to illustrate, not
to limit, the invention.
Preparation A
N,S-Diacyl Cysteamines
[0079] A. N,S-Diacetylcysteamine
[0080] Cysteamine hydrochloride (50.0 g) is added to a 1-L 3-neck
round bottom flask fitted with a magnetic stir bar, 2 addition
funnels, and a pH electrode. Water (300 ml) is added and the
stirred solution is cooled on ice. The pH is adjusted to 8.0 by
addition of 8 N KOH. Acetic anhydride (125 ml) is placed in one
addition funnel, and 8N KOH (350 ml) is placed in the other
addition funnel. The acetic anhydride is added dropwise to the
cysteamine solution, with 8 N KOH being added so as to keep the
reaction pH at 8 +/-1. After addition of acetic anhydride is
complete, the pH was adjusted to 7.0 using 1 N HCl and the mixture
is allowed to stir for 75 min on ice. Solid NaCl is added to
saturation, and the solution is extracted 4 times using 400 ml
portions of CH.sub.2Cl.sub.2. The organic extracts are combined,
dried over MgSO.sub.4, filtered, and concentrated under reduced
pressure to yield 68.9 g (97% yield) of a pale yellow oil, which
crystallizes upon standing at 4.degree. C. .sup.1H-NMR (CDCl.sub.3,
400 MHz): .delta.6.43 (br s,1H), 3.42 (q,2H,J=7), 3.03 (t,2H,J=7),
2.36 (s,3H), 1.98 (s,3H). .sup.13C-NMR (CDCl.sub.3, 100 MHz):
.delta.196.09, 170.45, 39.42, 30.56, 28.71, 23.06.
[0081] B. N,S-Dipropionylcysteamine
[0082] A solution of cysteamine hydrochloride (100 g) in 750 mL of
water in a 2-L round bottom flask fitted with a 250 ml addition
funnel and a magnetic stirrer was treated with potassium hydroxide
(49.4g). Sodium bicarbonate (222 g) was added after complete
dissolution of the KOH. The addition funnel was charged with
propionic anhydride (237 mL), which was added to the reaction over
a period of 1 hour. Upon completion of addition, the reaction was
stirred vigorously for an additional 1 hour. Solid sodium chloride
was added to saturation, and the solution was extracted 4 times
with 500 ml portions of CH.sub.2Cl.sub.2. The organic extracts were
combined, dried over MgSO.sub.4, filtered, and concentrated on
rotary evaporator to give 155.2 g (93% yield) of a pale yellow oil,
which crystallizes upon standing at 4.degree. C.; mp 48-49.degree.
C. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.5.8 (br s,1H); 3.44
(q,2H,J=6); 3.03 (t,2H,J=6); 2.59 (q,2H,J=7); 2.19 (q,2H,J=7); 1.18
(t,3H,J=7); 1.14 (t,3H,J=7). .sup.13C-NMR (CDCl.sub.3, 100 MHz):
.delta.200.64, 174.05, 39.45, 37.38, 29.52, 28.36, 9.74, 9.60.
[0083] C. N,S-dibutyrylcysteamine
[0084] Butyryl chloride (10.4 mL) was added dropwise to a solution
of cysteamine (3.86 g) and triethylamine (14 mL) in 150 mL of
dichloromethane at 0.degree. C. After addition, the mixture is
warmed to ambient temperature and stirred for an additional hour.
The mixture is poured into water, and the organic phase is
collected. The organics are washed sequentially with water, 1N HC1,
saturated NaHCO.sub.3, and brine, then dried over MgSO.sub.4,
filtered, and evaporated to yield a colorless oil. Crystallization
yields a waxy solid. .sup.1H-NMR (CDCl.sub.3): .delta.6.0 (br
s,1H), 3.44 (q,2H,J=6); 3.03 (t,2H,J=6); 2.55 (t,2H,J=7); 2.14
(t,2H,J=7); 1.67 (m,4H); 0.98 (t,3H,J=7); 0.94 (t,3H,J=7).
.sup.13C-NMR (CDCl.sub.3): .delta.200.00, 173.15, 45.86, 39.50,
38.52, 28.39, 19.09, 19.01, 13.66, 13.39.
[0085] D. N,S-dipentanoylcysteamine, N,S-dihexanoylcysteamine,
N,S-diheptanoylcysteamine, and N,S-dioctanoylcysteamine
[0086] These were prepared as in paragraph A by reaction of
cysteamine hydrochloride with the appropriate anhydride or acid
chloride.
Preparation B
Preparation of N-Acylcysteamines
[0087] A. N-Acetylcysteamine
[0088] N,S-diacetylcysteamine (42.64 g) is placed in a 2-L round
bottom flask fitted with a magnetic stirrer, and dissolved in 1400
ml of water. The flask is purged with N.sub.2, and the mixture is
chilled on an ice bath. Potassium hydroxide (49.42 g) is added, and
the mixture is stirred for 2 h on ice under inert atmosphere. The
pH is adjusted to 7 using 6 N HCl, and solid NaCl is added to
saturation. The mixture is extracted 7 times with 500 ml portions
of CH.sub.2Cl.sub.2. The organic extracts are combined, dried over
MgSO.sub.4, filtered, and concentrated under reduced pressure to
yield 30.2 g (96% yield) of product. This material is distilled
immediately prior to use, bp 138-140.degree. C./7 mmHg.
[0089] B. N-Propionylcysteamine
[0090] A solution of N,S-dipropionylcysteamine (18.9 g) in methanol
(100 mL) is placed under a nitrogen atmosphere with stirring. A
solution of sodium methoxide (25 wt %) in methanol (ca. 22 mL) is
added slowly until analysis by thin-layer chromatography (1:1 ethyl
acetate/hexane) reveals complete disappearance of starting
material. Oxalic acid dihydrate (6.3 g) is added, then the mixture
is vacuum filtered through a pad of Celite and evaporated to give a
colorless oil. Purification by distillation gives the product.
[0091] C. Additional N-acylcysteamines
[0092] Using the procedure of paragraph A, the corresponding
N-butyrylcysteamine, N-pentanoylcysteamine, hexanoylcysteamine,
N-heptanoylcysteamine, and N-octanoylcysteamine were prepared.
EXAMPLE 1
Preparation of (2S,3R)-2-methyl-3-hydroxyhexanoate
N-acetylcysteamine thioester
[0093] A.
(4S)-N-[(2S,3R)-2-methyl-3-hydroxyhexanoyl]-4-benzyl-2-oxazolidi-
none
[0094] A dry, 2 L three-necked round bottomed flask equipped with a
500 ml addition funnel, a low-temperature thermometer, and a stir
bar was charged with 19.84 g of N-propionyl-oxazolidinone, capped
with septa and flushed with nitrogen. Anhydrous dichloromethane
(100 ml) was added by cannula and the resulting solution was cooled
to -65.degree. C. in a bath of dry ice/isopropanol. The addition
funnel was charged by cannula with 100 ml of dibutylboron triflate
(1.0 M in dichloromethane), which was added in a slow stream to the
reaction. Triethylamine (15.6 ml) was added dropwise by syringe,
keeping the reaction temperature below -10.degree. C. The reaction
was then transferred to an ice bath and allowed to stir at
0.degree. C. for 30 minutes. After that period, the reaction was
placed back into the dry ice/isopropanol bath and allowed to cool
to -65.degree. C. Butyraldehyde (8.6 ml) was added rapidly by
syringe, and the reaction was allowed to stir for 30 min.
[0095] The reaction was transferred to an ice bath and the addition
funnel was charged with 100 ml of a 1 M aqueous phosphate solution,
pH 7.0 (the phosphate solution is comprised of equal molar amounts
of mono- and dibasic potassium phosphate). The phosphate solution
was added as quickly as possible while keeping the reaction
temperature, below 10.degree. C. The addition funnel was then
charged with 300 ml of methanol which was added as quickly as
possible while keeping the reaction temperature below 10.degree. C.
Finally, the addition funnel was charged with 300 ml of 2:1
methanol-30% hydrogen peroxide. This was added dropwise to ensure
that the temperature was kept below 10.degree. C. The reaction was
stirred for one hour after completion of addition. The solvent was
then removed on a rotary evaporator until a slurry remained. The
slurry was extracted 4 times with 500 ml portions of ethyl ether.
The combined organic extracts were washed with 250 ml each of
saturated aqueous sodium bicarbonate and brine. The extract was
then dried with MgSO.sub.4, filtered, and concentrated to give a
slightly yellow oil. The material was then chromatographed on
SiO.sub.2 using 2:1 hexanes:ethyl acetate (product Rf=0.4)
resulting in 22.0 g (85% yield) of title compound as a colorless
oil. APCI-MS: m/z 306 (MH+). .sup.1H-NMR (360 MHz, CDCl.sub.3):
.delta.7.2-7.4 (5H,m, phenyl); 4.71 (1H,m,H4); 4.17-4.25 (2H,m,H5);
3.96 (1H,m,H3'); 3.77 (1H,dq,J=2.5,7 Hz,H2'); 3.26 (1H,dd,J=4,13
Hz,benzylic); 2.79 (1H,dd,J=9,13 Hz,benzylic); 1.5-1.6 (2H,m,H4');
1.3-1.5 (2H,m,H5'); 1.27 (3H,d,J=7 Hz,2'-Me); 0.94 (3H,t,J=7
Hz,H6')
[0096] B. (2S,3R)-2-methyl-3-hydroxyhexanoate N-acetylcysteamine
thioester
[0097] N-acetylcysteamine was distilled at 130.degree. C./7 mmHg to
give a colorless liquid at room temperature. A dry, 1 L
three-necked round bottomed flask equipped with a 500 ml addition
funnel and a stir bar was capped with septa and flushed with
nitrogen. The flask was then charged with 10.7 ml of
N-acetylcysteamine by syringe and with 400 ml of anhydrous THF by
cannula. The mixture was cooled with a MeOH/ice bath. Butyllithium
(64 ml of 1.6 M in hexanes) was added dropwise by syringe,
resulting in formation of a white precipitate. After stirring for
30 min, trimethylaluminum (51 ml of 2.0 M in hexanes) was added
dropwise by syringe. The reaction became clear after addition of
trimethylaluminum and was allowed to stir an additional 30 min.
During this period, 20.5 g (0.068 mol) of
(4S)-N-[(2S,3R)-2-methyl-3-hydroxylhexanoyl]-4-benzyl-2-ox-
azolidinone was put under a blanket of nitrogen and dissolved in
100 ml of anhydrous THF; this solution was then transferred in a
slow stream by cannula into the reaction. The resulting reaction
mixture turned a yellow-green color and was allowed to stir for 1
hr. The reaction was judged complete when the starting material
could no longer be seen by thin-layer chromatographic analysis (ca.
1 hr).
[0098] The reaction was treated with enough saturated oxalic acid
to give a neutral reaction with pH paper (approximately 90 ml). The
solvents were then removed on a rotary evaporator to give a white
slurry. The slurry was extracted six times with 250 ml portions of
ethyl ether. The organic extracts were combined and washed with
brine, dried with MgSO.sub.4, filtered, and concentrated to give a
slightly yellow oil. The thioester product was purified by flash
chromatography on SiO.sub.2 using 1:1 hexanes:EtOAc until the
elution of 4-benzyl-2-oxazolidinone. At that point, the solvent
system was switched to 100% EtOAc to give pure fractions of
diketide thioester. The product fractions were combined and
concentrated to give 14.9 g (89% yield) of title compound. APCI-MS:
m/z 248 (MH+). .sup.1H-NMR (360 MHz, CDCl.sub.3): .delta.5.8 (br
s,1H); 3.94 (dt,1H), 3.46 (m,2H), 3.03 (dt,2H), 2.71 (dq,1H), 1.97
(s,3H), 1.50 (m,2H), 1.37 (m,2H), 1.21 (d,3H), 0.94 (t,3H).
[0099] C. In a manner similar to that set forth in paragraph A, but
substituting for N-acetylcysteamine, the various N-acylcysteamines
prepared in Preparation B, the corresponding N-acylcysteamine
thioesters of (2S,3R)-2-methyl-3-hydroxyhexanoate were
prepared.
EXAMPLE 2
Comparative Feeding of Diketide N-Acylcysteamine Thioesters
[0100] The N-acylcysteamine thioesters of
(.+-.)-(2S*,3R*)-2-methyl-3-hydr- oxy-hexanoate were fed to growing
cultures of Streptomyces coelicolor CH999/pJRJ2, and the production
of 15-methyl-6-deoxyerythronolide B was monitored. Duplicate
cultures were grown in 50 ml of medium (sucrose (103 g/l),
K.sub.2SO.sub.4 (0.25 g/l), MgCl.sub.2.6H.sub.2O (10.12 g/l),
casaminoacids (0.1 g/l), yeast extract (5 g/l), TES buffer (5.73
g/l), sodium propionate (10 mM), and trace elements) supplemented
with 50 ug/ml of thiostrepton. After 2 days post-inoculation, the
cultures were fed with a solution of diketide thioester in 9:1
water/DMSO to give a final concentration of 0.5 mM diketide
thioester. Aliquots of the cultures were removed periodically and
assayed for polyketide production by HPLC, with quantitation
performed by evaporative light scattering. Production of
15-methyl-6-dEB 6 days after feeding was as follows:
1 Yield of Acyl group 15-methyl-6-dEB Acetyl 26 mg/L Propionyl 35
mg/L Butyryl 30 mg/L Pentanoyl 35 mg/L Hexanoyl 33 mg/L Heptanoyl
27 mg/L Octanoyl 23 mg/L
[0101] These preliminary results indicate that relatively little
difference in yield is obtained depending on the acyl group coupled
to cysteamine, but that an optimum chain length at least with
respect to the diketide tested, is between 3-6C in the acyl
group.
[0102] The following examples 3-6 describe the preparation of
additional optically active forms of N-acylcysteamines
EXAMPLE 3
Preparation of (2S,3R)-2-methyl-3-hydroxy-4-pentenoate
N-acetylcysteamine thioester
[0103] A.
(4S)-N-[(2S,3R)-2-methyl-3-hydroxy-4-pentenoyl]-4-benzyl-2-oxazo-
lidinone
[0104] A dry, 2 L three-necked round bottomed flask equipped with a
500 ml addition funnel, a low-temperature thermometer, and a stir
bar was charged with 20.0 g of propionyl oxazolidinone A, capped
with septa and flushed with nitrogen. Anhydrous dichloromethane
(100 ml) was added and the resulting solution was cooled to
-15.degree. C. in a bath of methanol/ice. Dibutylboron triflate
(100 ml of 1.0 M in dichloromethane) was added in a slow stream via
the addition funnel at such a rate as to keep the reaction
temperature below 3.degree. C. Diisopropylethylamine (17.9 ml) was
added dropwise by syringe, again keeping the internal temperature
below 3.degree. C. The reaction was then cooled to -65.degree. C.
using a dry ice/isopropanol bath. Acrolein was added over 5 minutes
by syringe. The reaction was allowed to stir for 30 min after
completion of addition.
[0105] The reaction was then transferred to an ice bath and the
addition funnel was charged with 120 ml (0.1 mol) of a 1 M aqueous
phosphate solution, pH 7.0 (the phosphate solution is comprised of
equal molar amounts of mono- and dibasic phosphate). The phosphate
solution was added as quickly as possible while keeping the
reaction temperature below 10.degree. C. The addition funnel was
then charged with 400 ml methanol which was added as quickly as
possible while keeping the reaction temperature below 10.degree. C.
Finally, the addition funnel was charged with 400 ml of 2:1
methanol-30% hydrogen peroxide. This was added dropwise at first to
ensure that the temperature was kept below 10.degree. C. The
reaction was stirred for one hour. The solvent was then removed by
rotary evaporation until a slurry remained. The slurry was
extracted 4 times with 500 ml portions of ethyl ether. The organic
extracts were combined and washed with 250 ml each of saturated
sodium bicarbonate and brine, then dried with MgSO.sub.4, filtered,
and concentrated to give a slightly yellow oil. Trituration with
hexane induced crystallization. Recrystallization from ether by
addition of hexane resulted in 13.67 g (55% yield) of product.
.sup.1H-NMR (360 MHz, CDCl.sub.3): .delta.7.2-7.4 (m,5H); 5.86
(ddd,1H), 5.35 (dt,1H), 5.22 (dt,1H), 4.71 (m,1H), 4.51 (m,1H),
4.21 (m,2H), 3.89 (dq,1H), 3.26 (dd,1H), 2.80 (dd,1H), 1.25
(d,3H).
[0106] B. (2S,3R)-2-methyl-3-hydroxy-4-pentenoate
N-acetylcysteamine thioester
[0107] N-acetylcysteamine was distilled at 130.degree./7 mm to give
a colorless liquid at room temperature. A dry, 1 L three-necked
round bottomed flask equipped with a 500 ml addition funnel and a
stir bar was capped with septa and flushed with nitrogen. The flask
was then charged with 7.5 ml of N-acetylcysteamine by syringe and
with 500 ml of anhydrous THF by cannula. The reaction was then
cooled with a MeOH/ice bath. Butyllithium (44 ml of 1.6 M in
hexane) was added dropwise by syringe. A white precipitate formed
as the n-BuLi was added. After stirring for 30 min, 35.5 ml (0.071
mol) of trimethylaluminum (2.0 M in hexane) was added dropwise by
syringe. The reaction became clear after addition of
trimethylaluminum and was allowed to stir an additional 30 min.
(4S)-N-[(2S,3R)-2-methyl-3-hydroxy-4-pentenoyl]-4-benzyl-2-oxazolidinone
from paragraph A (13.6 g) was put under a blanket of nitrogen,
dissolved in 50 ml of anhydrous THF, and this solution was then
transferred in a slow stream by cannula into the reaction. The
resulting reaction mixture turned a yellow-green color and was
allowed to stir for 1 hr. The reaction was judged to be finished
when starting material could no longer be seen by thin-layer
chromatography (ca. 30 min).
[0108] Enough saturated oxalic acid was added to give a neutral
reaction with pH paper (approximately 60 ml). The solvents were
then removed by rotary evaporator to give a white slurry. The
slurry was extracted six times with 250 ml portions of ethyl ether
The organic extracts were combined, washed with brine, dried with
MgSO.sub.4, filtered, and concentrated to give a slightly yellow
oil. The thioester was then purified by flash chromatography on
SiO.sub.2. The column was run with 1:1 hexanes:ethyl acetate until
the elution of oxazolidinone. At that point, the eluent was
switched to 100% ethyl acetate to give pure fractions of product.
The fractions were combined and concentrated to give 7.7 g (71%
yield) of product. .sup.1H-NMR (360 MHz, CDCl.sub.3): .delta.5.82
(ddd,1H), 5.78 (br s, 1H), 5.32 (dt,1H), 5.21 (dt,1H), 4.47 (m,1H),
3.45 (m,2H), 3.04 (m,2H), 2.81 (dq,1H), 1.96 (s,3H), 1.22
(d,3H).
EXAMPLE 4
Preparation of (2S,3R)-2-methyl-3-hydroxy-4-pentnoate
N-acetylcysteamine thioester
[0109] A.
(4S)-N-[(2S,3R)-2-methyl-3-hydroxy-5-trimethylsilyl-4-pentvnoyl]-
-4-benzyl-2-oxazolidinone
[0110] Prepared according to the method of Example 1, paragraph A
by reaction of (4S)-N-propionyl-4-benzyl-2-oxazolidinone with
3-trimethylsilylpropargyl aldehyde in 80% yield.
[0111] B.
(4S)-N-[(2S,3R)-2-methyl-3-hydroxy-4-pentynoyl]-4-benzyl-2-oxazo-
lidinone
[0112] A solution of
(4S)-N-[(2S,3R)-2-methyl-3-hydroxy-5-trimethylsilyl-4-
-pentynoyl]-4-benzyl-2-oxazolidinone (0.13 g) in 3 mL of
dimethylformamide was treated with 48% aqueous HF (2.6 uL) and
KF.2H.sub.2O at ambient temperature for 100 min. Upon completion of
the reaction, saturated aqueous sodium bicarbonate was added to
neutralize the HF, and the mixture was extracted three times with
equal portions of ether. The organic extracts were combined,
filtered, and dried over MgSO4. Filtration and evaporation gave the
crude product, which was purified by silica gel chromatography (3:2
hexanes/ethyl acetate) to yield 64 mg of product.
[0113] C. (2S,3R)-2-methyl-3-hydroxy-4-pentynoate
N-acetylcysteamine thioester
[0114] In a 25 ml round bottom flask purged with N.sub.2, N-acetyl
cysteamine (0.12 ml, 1.1 mmol, 1.1 eq) was dissolved in 5.2 ml of
anhydrous THF. The solution was cooled to 0.degree. C. A 1.6 M
solution of butyllithium in hexanes (0.68 ml, 1.1 mmol, 1.1 eq) was
added with a syringe to give a heterogeneous mixture. A 2.0 M
solution of trimethylaluminum in hexanes (0.55 ml, 1.1 mmol, 1.1
eq) was added dropwise with vigorous stirring to give a
yellow-green solution. A solution of
(4S)-N-[(2S,3R)-2-methyl-3-hydroxyl-4-pentynoyl]-4-benzyl-2-o-
xazolidinone (280 mg, 1.0 mmol, 1.0 eq) in 2 ml of THE was added.
The solution was stirred for 15 min and neutralized with saturated
oxalic acid (aq). Volatiles were removed in vacuo. The resulting
slurry was extracted with 4.times.20 ml of ethyl acetate. The
combined extracts were washed with a minimum of saturated aqueous
CuSO.sub.4 to remove excess thiol. Some distilled water was used to
aid separation. The organic layer was dried over MgSO.sub.4,
filtered and concentrated. The resulting oil was purified by flash
chromatography to give 191 mg of title compound (83% yield) as a
pale yellow oil. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.5.76 (br
s,1H); 4.68 (dd,1H,J=2,4); 3.47 (m,2H); 3.05 (m,2H), 2.9 (dq,1H),
2.8 (br d,1H); 2.51 (d,1H,J=2); 1.97 (s,3H); 1.38 (d,3H,J=7).
[0115] D. Preparation of
(2S,3R)-5-fluoro-2-methyl-3-hydroxypentanoate N-acetylcysteamine
thioester
[0116] Prepared according to the procedure of Example 3, paragraph
B, from
(4S)-N-[(2S,3R)-5-fluoro-2-methyl-3-hydroxypentanoyl]-4-benzyl-2-oxazolid-
inone and N-acetylcysteamine. .sup.13C-NMR (100 MHz, CDCl.sub.3):
.delta.203.53, 170.65, 81.22 (d,J.sub.CF=163), 68.48
(d,J.sub.CF=4), 53.42, 39.12, 34.99 (d,J.sub.CF=26), 28.54, 23.07,
11.33.
EXAMPLE 5
Preparation of (4S,5R)-4-methyl-5-hydroxy-2-heptenoate
N-acetylcysteamine thioester
[0117] A.
(4S)-N-[(2S,4S,5R)-2,4-dimethyl-5-hydroxy-3-oxoheptanoyl]-4-benz-
yl-2-oxazolidinone
[0118] A solution of 2.0 g of
(4S)-N-[(2S)-2-methyl-3-oxopentanoyl]-4-benz- yl-2-oxazolidinone
(prepared according to the procedure of Evans, et al., Tetrahedron
(1992) 48:2127-2142) in 18 ml of CH.sub.2Cl.sub.2 was cooled to
-15.degree. C., and 0.89 ml of TiCl.sub.4 was added dropwise over 3
minutes, followed by addition of 1.38 ml of diisopropylethylamine
over 10 minutes. After stirring for 30 minutes, the mixture was
cooled to -78.degree. C. and 0.55 ml of propionaldehyde was added
over 20 minutes. The mix was stirred overnight, then quenched with
20 ml of saturated NH.sub.4Cl and allowed to warm to ambient
temperature. Water (5 ml) was added, and the resulting mixture was
extracted three times with 75 ml portions of ether. The organic
extracts were combined, washed with saturated NH.sub.4Cl, saturated
NaHCO.sub.3, and brine, then dried over MgSO.sub.4 and
concentrated. The crude product was purified by chromatography on
SiO.sub.2 using a gradient from 9:1 to 1:1 hexanes/ethyl acetate,
yielding 1.9 gm (79%) of the product.
[0119] B.
(4S)-N-[(2S,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl]-4-ben-
zyl-2-oxazolidinone
[0120] Tetramethylammonium triacetoxyborohydride (2.89 g) was
dissolved in a mixture of acetic acid (11 ml) and acetonitrile (11
ml), stirred for 30 minutes at ambient temperature, then cooled to
-15 .degree.C. before addition of
(4S)-N-[(2S,4S,5R)-2,4-dimethyl-5-hydroxy-3-oxoheptanoyl]-4-b-
enzyl-2-oxazolidinone (0.764 g). After stirring for 4 hours, 34 ml
of 0.5 M sodium tartrate was added and stirring was continued for
an additional 3 hours. After extraction with 3 portions of
CH.sub.2Cl.sub.2, the organic phases were combined and dried over
MgSO.sub.4. The solvent was removed under vacuum, and the crude
product was evaporated 3 times from 50 ml of methanol to yield
0.644 g of product (84%).
[0121] C.
(4S)-N-[(2S,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl]-4-ben-
zyl-2-oxazolidinone 3',5'-cyclic carbonate Triphosgene (0.138 g)
was added to a -15.degree. C. solution of
(4S)-N-[(2S,3S,4S,5R)-2,4-dimethyl-3,5-di-
hydroxyheptanoyl]-4-benzyl-2-oxazolidinone (0.175 g),
diisopropylethylamine (0.52 ml) and 4-dimethylaminopyridine (0.02
g) in 2 ml of CH.sub.2Cl.sub.2. After stirring for 16 hours, the
reaction was quenched by addition of 2 ml of sat. NH.sub.4Cl and
was extracted with ethyl acetate. The organic extract was washed
with sat. NH.sub.4Cl and brine, then concentrated to give an orange
oil. Chromatography (SiO.sub.2) gave the pure cyclic carbonate (71
mg).
[0122] D.
(4S)-N-[(4S,5R)-4-methyl-5-hydroxy-2-heptenoyl]-4-benzyl-2-oxazo-
lidinone
[0123] A solution of
(4S)-N-[(2S,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxyhepta-
noyl]-4-benzyl-2-oxazolidinone 3',5'-cyclic carbonate (71 mg) in 4
ml of tetrahydrofuran was treated with 0.052 ml of
diazabicycloundecene at ambient temperature for 16 hours. Addition
of 5 ml of sat. NH.sub.4Cl followed by extraction with ethyl
acetate and evaporation of solvent yielded crude product, which was
chromatographed on SiO.sub.2 to give pure material (31 mg,
50%).
[0124] E. (4S,5R)-4-methyl-5-hydroxy-2-heptenoate
N-acetylcysteamine thioester
[0125] A solution of N-acetylcysteamine (0.064 ml) in 3.4 ml of
tetrahydrofuran at -15.degree. C. was treated with 0.38 ml of 1.6 M
n-butyllithium in hexanes followed by 0.30 ml of 2.0 M
trimethylaluminum in hexanes and stirred for 30 minutes. A 0.54 ml
portion of this solution was then added to a solution of 31 mg of
(4S)-N-[(4S,5R)-4-methyl-5-hydro-
xy-2-heptenoyl]-4-benzyl-2-oxazolidinone in 0.3 ml of
tetrahydrofuran and the mixture was stirred for 2 hours before
neutralization with saturated aqueous oxalic acid.
EXAMPLE 6
Additional Precursors to Optically Active N-Acyl Cysteamine
Thioesters
[0126] A. Preparation of
(4S)-N-[(2S,3R)-2-methyl-3-hydroxybutanoyl]-4-ben-
zyl-2-oxazolidinone
[0127] Prepared from (4S)-N-propionyl-4-benzyl-2-oxazolidinone and
acetaldehyde according to the procedure described in Example 1,
paragraph A. .sup.1H-NMR (360 MHz, CDCl.sub.3): .delta.7.2-7.4
(m,5H); 4.71 (m,1H); 4.12-4.25 (m,2H); 3.76 (dq,1H); 3.26 (dd,1H);
2.79 (dd,1H); 1.30 (d,3H), 1.21 (d,3H).
[0128] B. Preparation of
(4S)-N-[(2S,3R)-2-vinyl-3-hydroxypentanoyl]-4-ben-
zyl-2-oxazolidinone
[0129] A solution of 2.45 g of
(4S)-N-crotonyl-4-benzyl-2-oxazolidinone in 10 ml of anhydrous
CH.sub.2Cl.sub.2 was cooled to -78.degree. C., and 1.7 ml of
triethylamine was added followed by 10.5 ml of a 1 M solution of
dibutylboron triflate in CH.sub.2Cl.sub.2. After 30 minutes, the
reaction was warmed to 0.degree. C., kept for 20 minutes, then
recooled to -78.degree. C. Propionaldehyde (0.9 ml) was added, and
the reaction was allowed to slowly warm to ambient temperature over
16 hours. Standard oxidative workup yielded the product (1.98 g,
65% yield) after chromatography (2:1 hexane/ethyl acetate).
.sup.1H-NMR (360 MHz, CDCl.sub.3): .delta.7.2-7.35 (m,5H); 6.02
(1H,m); 5.41 (m,2H); 4.72 (m,1H); 4.58 (dd,1H); 4.20 (m,2H); 3.92
(m,1H); 3.25 (dd,1H); 2.98 (br s, 1H); 2.76 (dd,1H); 1.53 (m,2H);
0.98 (t,3H).
[0130] C. Preparation of
(4S)-N-[(2S,3R)-2-methyl-3-hydroxy-3-(3-pyridyl)p-
ropanoyl]-4-benzyl-2-oxazolidinone 12
[0131] Prepared from (4S)-N-propionyl-4-benzyl-2-oxazolidinone and
pyridine-3-carboxaldehyde according to the procedure described in
Example 1, paragraph A. .sup.13C-NMR (100 MHz, CDCl.sub.3):
.delta.176.40, 152.83, 148.74, 147.85, 136.78, 134.80, 134.04,
129.34, 128.95, 127.45, 123.21, 109.75, 71.46, 66.23, 55.05, 44.28,
37.70, 10.64.
[0132] D.
(4S)-N-[(2S,3R)-5-fluoro-3-hydroxy-2-methylpentanoyl)]-4-benzyl--
2-oxazolidinone 13
[0133] (a) A solution of 3-fluoropropanol in dichloromethane was
oxidized with the Dess-Martin periodinane. Analysis by .sup.1H-NMR
revealed complete oxidation to 3-fluoropropanal. The suspension was
filtered, washed with saturated sodium thiosulfate, then dried over
MgSO.sub.4. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.9.8 (t,1H),
4.8 (dt,2H), 2.85 (dt,2H).
[0134] (b) The aldol adduct was prepared according to the method of
Example 1, paragraph A by reaction of
(4S)-N-propionyl-4-benzyl-2-oxazoli- dinone with the solution of
3-fluoropropanal. .sup.13C-NMR (100 MHz, CDCl.sub.3):
.delta.177.08,153.00,134.94, 129.37, 128.91, 127.39, 81.16
(d,J.sub.CF=163), 67.67 (d,J.sub.CF=4), 66.20, 55.08, 42.23, 37.71,
34.52 (d,J.sub.CF=19), 10.68.
[0135] Examples 7-14 illustrate the preparation of racemic diketide
thioesters.
EXAMPLE 7
Preparation of 2-Benzoxazolone and Chlorozoxazone Intermediates
[0136] 14
[0137] A. N-propionyl-2-benzoxazolone
[0138] A solution of 135 g of 2-benzoxazolone (1.0 mol) in 750 mL
of acetone was treated with 14 g (0.1 mol) of potassium carbonate
and with 130 mL (1.0 mol) of propionic anhydride at ambient
temperature with stirring. After 4 hours, the mixture was poured
into 3000 mL of water with vigorous stirring. The precipitated
product was collected by vacuum filtration, washed with water, and
air dried to yield 187 g (98%) of light tan-colored product
suitable for further use; mp =88-90 .degree. C. (uncorr).
Recrystallization from ether/hexane yields the pure product, 172 g
(90% yield), mp=92-93.degree. C. (uncorr). .sup.1H-NMR (CDCl.sub.3,
400 MHz): .delta.8.07 (1H,m); 7.21 (1H,m); 7.22-7.28 (2H,m); 3.12
(2H,q,J=7 Hz); 1.28 (3H,t,J=7 Hz). .sup.13C-NMR (CDCl.sub.3, 100
MHz): .delta.173.3, 151.3, 142.2, 127.8, 125.1, 124.7, 115.9,
109.7, 30.4, 7.9.
[0139] B. N-propionylchlorzoxazone 15
[0140] A solution of 17 g of chlorzoxazone
(5-chloro-2-benzoxazolone) (0.1 mol) in 75 mL of acetone was
treated with 1.0 g (0.007 mol) of potassium carbonate and 15 mL
(0.12 mol) of propionic anhydride at ambient temperature with
stirring. After 4 hours, the mixture was poured into 300 mL of
water with vigorous stirring. The precipitated product was
collected by vacuum filtration, washed with water, and air dried to
yield 22 g (98%) of colorless product; mp=97-99.degree. C.
(uncorr). .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.8.11 (d, 1H,
J=2 Hz); 7.23 (dd, 1H, J=2,9 Hz); 7.13 (d, 1H, J=9 Hz); 3.12 (q,
2H, J=7 Hz); 1.28 (t, 3H, J=7 Hz). .sup.13C-NMR (CDCl.sub.3, 100
MHz): .delta.173.08, 150.97, 140.67, 130.29, 128.45, 125.16,
116.41, 110.59, 30.41, 7.89.
[0141] C. (.+-.)-N-[(2R*,
3S*)-(2-methyl-3-hydroxyhexanoyl)]-2-benzoxazolo- ne 16
[0142] A solution of N-propionyl-2-benzoxazolone (100.0 g) in
anhydrous CH.sub.2Cl.sub.2 (1100 mL) was cooled to 3.degree. C.
with mechanical stirring under N.sub.2 atmosphere. TiCl.sub.4 (58.4
mL) was added at a rate such that the internal temperature remained
below 10.degree. C. (ca. 10 minutes). The resulting yellow slurry
was stirred vigorously for 40 minutes, then triethylamine (87.4 mL)
was added at a rate such that the internal temperature remained
below 10.degree. C. (ca. 10 minutes). The resulting deep red
solution was stirred for 80 minutes. Butyraldehyde (58.9 mL) was
added at a rate such that the internal temperature remained below
10.degree. C. (ca. 20 minutes), and the reaction was followed by
thin-layer chromatography (4:1 hexanes/ethyl acetate). After
stirring for 90 minutes, the reaction was quenched by addition of
450 mL of 2 N HCl. The phases were separated, and the aqueous phase
was extracted 3 times with 750-mL portions of ether. The organic
phases were combined and washed three times with 200-mL portions of
2 N HCl. The acidic washes were combined and back-extracted 3 times
with 150-mL portions of ether. The combined organic extract was
washed once with 300 mL of sat. aq. NaHCO.sub.3, and once with 300
mL of sat. aq. NaCl. The organic phase was then dried over
MgSO.sub.4, filtered, and concentrated under vacuum to a yellow
slurry. The product was collected by vacuum filtration and rinsed
with hexanes to yield a colorless solid. Concentration of the
filtrate yielded a second crop of product, which was collected in
the same manner, giving a combined 103 g (80% yield) of crystalline
product; mp=123-4.degree. C. The mother liquor can be
chromatographed (4:1 hexanes/ethyl acetate) to yield additional
product. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.8.10 (1H,m),
7.23-7.32 (3H,m), 4.12 (1H,m), 3.98 (1H,dq,J=3, 7), 2.26 (1H, br
s), 1.38-1.64 (4H,m), 1.34 (3H,d,J=7), 0.98 (3H,t,J=7).
.sup.13C-NMR (CDCl.sub.3, 100 MHz): .delta.176.4, 151.1, 142.2,
127.8, 125.5, 124.9, 1116.3, 109.9, 71.3, 43.7, 36.2, 19.2, 13.9,
10.1.
[0143] D.
(.+-.)-N-[(2R*,3S*)-(2-methyl-3-hydroxy-4-pentenoyl)]-2-benzoxaz-
olone 17
[0144] This compound was prepared according to the procedure of
paragraph C, by reaction of N-propionyl-2-benzoxazolone with
acrolein. .sup.1H-NMR (CDCl.sub.3, 400 MHz) .delta.8.06 (m, 1 H),
7.27-7.20 (m, 2 H), 5.91 (ddd, J=17, 10, 5 Hz). 5.37 (dt, J=1, 17
Hz, 1 H), 5.24 (dt, J=1, 10 Hz, 1 H), 4.60 (m, 1 H), 4.06 (dq, J=3,
6 Hz, 1 H), 2.62 (d, J=4 Hz, 1 H), 1.30 (d, 6 Hz, 3 H).
.sup.13C-NMR (CDCl.sub.3, 100 MHz) .delta.175.4, 151.1, 142.2,
137.2, 127.7, 125.5, 124.9, 116.6, 116.2, 109.9, 72.7, 44.0,
10.7.
[0145] E.
(.+-.)-N-[(2R*,3S*)-(2-methyl-3-hydroxyheptanoyl)]-2-benzoxazolo-
ne 18
[0146] This compound was prepared according to the method of
paragraph C by reaction of N-propionyl-2-benzoxazolinone with
pentanal.
[0147] F.
(.+-.)-N-[(2R*,3S*)-(2-methyl-3-hydroxy-6-heptenoyl)]-2-benzoxaz-
olone 19
[0148] Was prepared according to the method of paragraph C by
reaction of N-propionyl-2-benzoxazolinone with 4-pentenal.
.sup.13C-NMR (CDCl.sub.3, 100 MHz): .delta.176.28, 151.07, 142.20,
137.96, 127.75, 125.50, 124.89, 116.26, 115.19, 109.89,70.93,43.76,
33.12, 30.16, 10.21.
[0149] G.
(.+-.)-N-[(2R*,3S*)-(2-methyl-3-hydroxyoctanoyl)]-2-benzoxazolon- e
20
[0150] Was prepared according to the method of paragraph C by
reaction of N-propionyl-2-benzoxazolinone with hexanal.
[0151] H.
(.+-.)-N-[(2R*,3S*)-(2,5-dimethyl-3-hydroxyhexanoyl)]-2-benzoxaz-
olone 21
[0152] Prepared according to the method of paragraph C by reaction
of N-propionyl-2-benzoxazolinone with 3-methylbutanal. .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta.8.06 (m,1 H); 7.25 (m,3H); 4.17
(m,1H); 3.91 (dq,1H,J=3,7); 2.52 (br s,1H); 1.82 (m,1H); 1.56
(ddd,1H,J=5,9,13); 1.31 (d,3H,J=7); 1.25 (ddd,1H,J=4,6,13); 0.95
(d,3H,J=7); 0.94 (d,3H,J=7).
[0153] I.
(.+-.)-N-[(2R*,3S*)-(2-methyl-3-hydroxy-5-phenylpentanoyl)]-2-be-
nzoxazolone 22
[0154] Prepared according to the method of paragraph C by reaction
of N-propionyl-2-benzoxazolinone with 3-phenylpropanal. .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta.8.06 (m,1H); 7.25 (m,8H); 4.11
(dt,1H,J=4,7); 3.96 (dq,1H,J=3,7); 2.91 (m,1H); 2.70 (m,1H); 1.95
(m,1H); 1.81 (m,1H); 1.34 (t,3H,J=7).
[0155] J.
(.+-.)-N-[(2R*,3S*)-(4-(2-methoxyethoxy)-2-methy-3-hydroxybutano-
yl)]-2-benzoxazolone 23
[0156] Prepared according to the method of paragraph C by reaction
of N-propionyl-2-benzoxazolinone with
(2-methoxyethoxy)acetaldehyde. .sup.13C-NMR (CDCl.sub.3, 100 MHz):
.delta.175.01, 151.02, 142.18, 127.91, 125.30, 124.77, 116.15,
109.76, 73.23, 71.86, 70.73, 70.64, 58.88, 41.63, 11.88.
[0157] K.
(.+-.)-N-[(2R*,3S*)-(2-methyl-3-hydroxy-3-phenylpropanoyl)]-2-be-
nzoxazolone 24
[0158] Prepared according to the method of paragraph C by reaction
of N-propionyl-2-benzoxazolinone with benzaldehyde; mp
155-158.degree. C. .sup.1NMR (CDCl.sub.3) .delta.8.10 (m, 1H), 7.45
(m, 2H), 7.35 (m, 2H), 7.26 (m, 4H), 5.30 (d, 1H), 4.26 (dq, J=3, 6
Hz, 1H), 1.26 (d, 3H). .sup.13C NMR (CDCl.sub.3) .delta.175.6,
151.0, 142.2, 141.0, 128.4, 127.7, 126.0, 125.5, 124.9, 116.3,
110.0, 73.2, 46.0, 10.3.
[0159] L.
(.+-.)-N-[(2R*,3S*)-(5-azido-2-methyl-3-hydroxypentanoyl)]-2-ben-
zoxazolone 25
[0160] (a) 3-azidopropanal was prepared by addition of HN.sub.3 to
acrolein according to A. J. Davies, et al. (1967) J. Chem. Soc.,
2109-2112, and gave the following NMR data: .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta.9.80 (t, 1H, J=1 Hz); 3.61 (t, 2H,
J=7 Hz); 2.74 (dt, 2H, J=1,7 Hz). .sup.13.sup.3C-NMR (CDCl.sub.3,
400 MHz): .delta.199.41, 44.42, 42.70.
[0161] (b) The aldol adduct of 3-azidopropanal. and
N-propionyl-2-benzoxazolone was prepared according to the procedure
of paragraph C. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.8.07 (m,
1H); 7.26 (m,3H); 4.23 (dq, 1H, J=3,10 Hz); 3.96 (dq, 1H, J=3, 7
Hz); 3.52 (dd, 2H, J=6, 8 Hz); 2.80 (dd, 1H, J=1,3 Hz); 1.84 (m,
2H); 1.75 (m, 2H); 1.34 (d, 3H, J=7 Hz). .sup.13C-NMR (CDCl.sub.3,
100 MHz): .delta.176.05, 151.07, 142.20, 127.63, 125.63, 124.97,
116.26, 109.96, 68.85, 48.44, 43.80, 32.94, 10.48.
[0162] M.
(.+-.)-N-[(2R*,3S*)-(5-chloro-2-methyl-3-hydroxypentanoyl)]-2-be-
nzoxazolone 26
[0163] (a) A solution of 3-chloropropanal in CH.sub.2Cl.sub.2 was
prepared by addition of HCl to acrolein according to the procedure
described above for 3-bromopropanal, and gave the following NMR
data: .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.9.78 (t, 1H, J=1
Hz); 3.80 (t, 2H, J=7 Hz); 2.93 (dt, 2H, J=1,7 Hz). .sup.13C-NMR
(CDCl.sub.3, 100 MHz): .delta.198.77, 45.90, 36.79.
[0164] (b) This solution was reacted with
N-propionyl-2-benzoxazolone according to the procedure of paragraph
C to yield the product, which was crystallized from ether/hexane;
mp=116-7.degree. C. .sup.13C-NMR (CDCl.sub.3, 100 MHz):
.delta.176.12, 151.06, 142.19, 127.63, 125.63, 124.96, 116.25,
109.96, 68.40, 43.68, 41.70, 36.46, 10.55.
[0165] N.
(.+-.)-N-[(2R*,3S*)-(5-(2-pyrimidinylthio)-2-methyl-3-hydroxypen-
tanoyl)]-2-benzoxazolone 27
[0166] (a) A suspension of 2-mercaptopyrimidine (6 g, 52 mmol) in
ethyl acetate (25 mL) was treated with 4 mL of acrolein and 100 mg
of tetrabutylammonium hydroxide at 70.degree. C. The bright yellow
suspension turned orange and cleared noticeably. After 30 min, the
mixture was cooled, filtered, and evaporated to yield 8.53 gm (95%)
of the product as an orange oil. .sup.1H-NMR (CDCl.sub.3, 400 MHz):
.delta.9.83 (t, 1H, J=1 Hz); 8.51 (d, 2H, J=5 Hz); 7.00 (t, 1H, J=5
Hz); 3.40 (t, 2H, J=7 Hz); 2.97 (dt, 2H, J=1, 7 Hz). .sup.13C-NMR
(CDCl.sub.3, 400 MHz): .delta.200.51, 171.69, 157.30, 116.66,
43.67, 23.31.
[0167] (b) The aldol adduct between 3-(2-pyrimidinyl-thio)propanal
and N-propionyl-2-benzoxazolone was prepared according to the
procedure of paragraph C. .sup.1H-NMR (CDCl.sub.3, 400 MHz):
.delta.8.49 (d, 2H, J=5 Hz); 8.07 (m, 1H); 7.25 (m, 3H); 6.97
(t,1H, 5 Hz); 4.25 (m, 1H); 4.01 (dq, 1H, J=3, 7 Hz); 3.92 (br d,
1H, J=4 Hz); 3.32 (m, 2H); 2.05 (m, 1H); 1.95 (m, 1H); 1.35 (d, 3H,
J=7 Hz). .sup.13C-NMR (CDCl.sub.3, 100 MHz): .delta.175.58, 172.66,
157.28, 151.04, 142.16, 127.81, 125.38, 124.81, 116.51, 116.22,
109.81, 69.62, 43.87, 34.29, 27.34, 10.89.
[0168] O
(.gamma.)-N-[(2R*,3S*)-(3-hydroxy-2,4,4-trimethylpentanoyl)]-2-be-
nzoxazolone 28
[0169] Prepared according to the method of paragraph C by reaction
of N-propionyl-2-benzoxazolone with trimethylacetaldehyde. Slow
addition of the aldehyde to the enolate solution over 1 hour at
0.degree. C. gave a 9:1 ratio of the (2R*,3S*) and (2R*,3R*)
isomers. The desired isomer was crystallized from 1:1
ether/hexanes, mp=90-2.degree.C. .sup.1H-NMR (CDCl.sub.3, 400 MHz)
.delta.8.04 (m, 1H), 7.23-7.29 (m, 3 H), 4.32 (dq,J=3,7 Hz, 1H),
3.79 (d,J=10 Hz, 1H), 3.41 (dd, J=3,10 Hz, 1 H), 1.51 (d,J=7 Hz,
3H), 0.94 (s, 9 H). .sup.13C-NMR (CDCl.sub.3, 100 MHz)
.delta.178.2, 150.8, 142.0, 127.6, 125.7, 125.0, 116.3, 110.0,
84.0, 37.4, 36.2, 26.7, 18.1.
[0170] P.
(.+-.)-N-[(2R*,3R*)-(3-hydroxy-2,4,4-trimethylpentanoyl)]-2-benz-
oxazolone 29
[0171] Prepared according to the method of paragraph C by reaction
of N-propionyl-2-benzoxazolone with trimethylacetaldehyde. Rapid
addition of the aldehyde to the enolate solution at 0.degree. C.
gave a 1:1 ratio of the (2R*,3S*) and (2R *,3R*) isomers. The
desired isomer was isolated by silica gel chromatography, then
crystallized. .sub.1H-NMR (CDCl.sub.3, 400 MHz) .delta.8.05 (m, 1
H), 7.20-7.27 (m, 4 H), 4.24 (dq, J=7, 4 Hz, 1 H), 3.82 (br t, J=4
Hz, 1 H), 2.33 (br d, J=4 Hz, 1 H), 1.36 (d, J=7 Hz, 3 H), 0.99,
(s, 9 H). .sup.13C-NMR (CDCl.sub.3, 100 MHz) .delta.175.3, 150.8,
142.2, 127.9, 125.4, 124.9, 116.2, 109.9, 77.6, 40.3, 35.8, 29.7,
26.7, 12.7.
[0172] Q.
(.+-.)-N-[(2R*,3S*)-4-benzyloxy-3-hydroxy-2-methylbutanoyl)]-2-b-
enzoxazolone and
(.+-.)-N-[(2R*,3R*)-4-benzloxy-3-hydroxy-2-methylbutanoyl-
)]-2-benzoxazolone 30
[0173] Prepared according to the procedure of paragraph C, by
reaction of N-propionyl-2-benzoxazolone with benzyloxyacetaldehyde.
The reaction yielded a 9:1 mixture of (2R*,3S*) and (2R*,3R*)
isomers. The isomers were separated by silica gel chromatography:
(2R*,3S*): .sup.1H-NMR (CDCl.sub.3, 400 MHz) .delta.8.03 (m, 1 H),
7.20-7.33 (m, 9 H), 4.55 (dd, J=25, 8 Hz, 2 H), 4.26 (br q, J=5 Hz,
1 H), 4.10 (dq, J=5, 6 Hz, 1 H), 3.59 (m, 2 H), 1.36 (d, 7 Hz, 3
H). .sup.13C-NMR (CDCl.sub.3,100 MHz) .delta.175.2, 151.0, 142.2,
137.6, 128.4(2), 127.7(2), 125.4, 124.8, 116.2, 109.8, 7304, 71.6,
70.7, 41.7, 11.9. (2R*,3R*): .sup.1H-NMR (CDCl.sub.3, 400 MHz)
.delta.8.02 (m, 1 H), 7.16-7.33 (m, 9 H), 4.52 (q,J=12 Hz, 2 H),
4.28 (p, J=7 Hz, 1 H), 4.04 (br m, 1 H), 3.69 (dd, J=3, 10 Hz, 1
H), 3.64 (dd, J=5, 10 Hz, 1 H), 3.09 (br s, 1 H), 1.31 (d, J=7H Hz,
3 H). .sup.13C-NMR (CDCl.sub.3, 100 MHz) .delta.175.8, 151.3,
142.0, 137.5, 128.3, 127.8, 127.7, 127.5, 125.3, 124.7, 116.2,
109.7, 73.5, 73.4, 73.0, 41.1, 14.5.
[0174] R. (.+-.)-N-[(2R*,3S*)-3-hydroxy
-2-methly-4-hexenoyl)]-2-benzoxazo- lone 31
[0175] Prepared according to the method of paragraph C by reaction
of N-propionyl-2-benzoxazolone with trans-crotonaldehyde; mp
74-6.degree. C. .sup.1H NMR (CDCl.sub.3) .delta.8.06 (m, 1 H); 7.23
(m, 3 H); 5.78 (dqd, 1 H,J=15, 7, 1 Hz); 5.55 (ddq, 1 H,J=15, 7, 2
Hz); 4.52 (br, 1 H); 4.05 (qd, 1 H, J=7, 4 Hz); 2.38 (br d, 1 H,
J=3 Hz); 1.70 (ddd, 3 H,J=7, 1, 1 Hz, 3 H); 1.30 (d, 3 H, J=7 Hz).
.sup.13C NMR (CDCl.sub.3) .delta.175.50, 151.20, 142.18, 129.99,
128.78, 127.80, 125.43, 124.86, 116.22, 109.85, 72.92, 44.31,
17.71, 11.04.
[0176] S.
(.+-.)-N-(2-(1-hydroxycyclohexyl)propionyl)-2-benzoxazolone 32
[0177] Prepared according to the method of paragraph C by reaction
of N-propionyl-2-benzoxazolone with cyclohexanone; mp 58-9.degree.
C. .sup.1H NMR (CDCl.sub.3) .delta.8.09 (m, 1 H); 7.25 (m, 3 H);
4.11 (q, 1 H, J=7 Hz); 2.90 (br s, 1 H); 1.82 (br d, 1 H, J=13 Hz);
1.59 (m, 8 H); 1.34 (d, 3 H, J=7 Hz); 1.23 (m, 1 H). .sup.13C NMR
(CDCl.sub.3) .delta.177.20, 151.42, 142.03, 127.69, 125.55, 124.91,
116.38, 109.88, 72.89, 46.77, 37.03, 33.37, 25.64, 21.73, 21.40,
12.16.
[0178] T.
(.+-.)-N-[(2R*,3S*)-6-benzyloxy-3-hydroxy-2-methylhexanoyl)]-2-b-
enzoxazolone 33
[0179] Is prepared according to the method of paragraph C by
reaction of N-propionyl-2-benzoxazolone with
4-benzyloxybutyraldehyde.
[0180] U.
(.+-.)-N-[(2R*,3S*)-6,6,6-trifluoro-3-hydroxy-2-methylhexanoyl)]-
-2-benzoxazolone 34
[0181] Prepared according to the method of paragraph C by reaction
of N-propionyl-2-benzoxazolone with 4,4,4-trifluorobutyraldehyde.
.sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.8.05 (m,1H); 7.25 (m,3H);
4.10 (dt,1H,J=3,10); 3.95 (dq,1H,J=3,7); 2.65 (br s,1H); 2.43
(m,1H); 2.17 (m,1H); 1.76 (m,2H); 1.33 (d,3H,J=7). .sup.19F-NMR
(CDCl.sub.3, 386 MHz): .delta.-66.77.
[0182] V.
(.+-.)-N-[(2R*,3S*)-5-methylthio-3-hydroxy-2-methylpentanoyl)]-2-
-benzoxazolone 35
[0183] Prepared according to the method of paragraph C by reaction
of N-propionyl-2-benzoxazolone with 3-(methylthio)propionaldehyde.
.sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.8.06 (m,1H); 7.25 (m,3H);
4.25 (m,1H); 3.96 (dq,1H,J=3,7); 2.82 (br d,1H) 2.68 (m,2H); 2.11
(s,3H); 1.90 (m,1H); 1.78 (m,1H); 1.34 (d,3H,J=7).
[0184] W.
(.+-.)-N-[(2R*,3S*)-4-cyclohexyl-3-hydroxy-2-methylbutanoyl)]-2--
benzoxazolone 36
[0185] Prepared according to the method of paragraph C by reaction
of N-propionyl-2-benzoxazolone with cyclohexylacetaldehyde.
[0186] X.
(.+-.)-N-[(2R*,3S*)-5-(3-pyridyl)-3-hydroxy-2-methylpentanoyl)]--
2-benzoxazolone 37
[0187] Is prepared according to the method of paragraph C by
reaction of N-propionyl-2-benzoxazolone with
3-(3-pyridyl)propanal.
[0188] Y.
(.+-.)-N-[(2R*,3S*)-3-hydroxy-2-methyl-5-hexenoyl)]-2-benzoxazol-
one 38
[0189] Is prepared according to the method of paragraph C by
reaction of N-propionyl-2-benzoxazolone with 3-butenal.
[0190] Z.
(.+-.)-N-[(2R*,3S*)-4-methoxy-3-hydroxy-2-methylbutanoyl)]-2-ben-
zoxazolone 39
[0191] Is prepared according to the method of paragraph C by
reaction of N-propionyl-2-benzoxazolone with
methoxyacetaldehyde.
[0192] AA.
(.+-.)-N-[(2R*,3S*)-3-(2-methylthiazol-4-yl)-3-hydroxy-2-methyl-
propanoyl)]-2-benzoxazolone 40
[0193] Is prepared according to the method of paragraph C by
reaction of N-propionyl-2-benzoxazolone with
2-methylthiazole-4-carboxaldehyde.
[0194] BB.
(.+-.)-N-[(2R*,3S*)-5-(2-methylthiazol-4-yl)-3-hydroxy-2-methyl-
pentanoyl)]-2-benzoxazolone 41
[0195] Is prepared according to the method of paragraph C by
reaction of N-propionyl-2-benzoxazolone with
3-(2-methylthiazol-4-yl)propanal.
[0196] CC.
(.+-.)-N-[(2R*,3*)-3-hydroxy-2-methyl-5-heptynoyl)1-2-benzoxazo-
lone 42
[0197] Is prepared according to the method of paragraph C by
reaction of N-propionyl-2-benzoxazolone with 3-pentynal.
[0198] DD.
(.+-.)-N-[(2R*,3S*)-3-(tetrahydrofuran-2-yl)-3-hydroxy-2-methyl-
propanoyl)]-2-benzoxazolone 43
[0199] Is prepared according to the method of paragraph C by
reaction of N-propionyl-2-benzoxazolone with
tetrahydrofuran-2-carboxaldehyde.
[0200] EE.
(.+-.)-N-[(2R*,3S*)-5-(methoxycarbonyl)-3-hydroxy-2-methylpenta-
noyl)]-2-benzoxazolone 44
[0201] Is prepared according to the method of paragraph C by
reaction of N-propionyl-2-benzoxazolone with methyl
4-oxobutanoate.
[0202] FF.
(.+-.)-N-f(2R*,3S*)-5-fluoro-3-hydroxy-2-methylpentanoyl)]-2-be-
nzoxazolone 45
[0203] (a) A solution of 3-fluoropropanol in dichloromethane was
oxidized with the Dess-Martin periodinane. Analysis by .sup.1H-NMR
revealed complete oxidation to 3-fluoropropanal. The suspension was
filtered, washed with saturated sodium thiosulfate, then dried over
MgSO.sub.4. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.9.8 (t,1 H),
4.8 (dt,2H), 2.85 (dt,2H).
[0204] (b) The aldol adduct is prepared according to the method of
paragraph C by reaction of N-propionyl-2-benzoxazolone with the
solution of 3-fluoropropanal.
[0205] GG.
(.+-.)-N-[(2R*,3S*)-(5-phthalimido-2-methyl-3-hydroxypentanoyl)-
]-2-benzoxazolone 46
[0206] (a) 3-Phthalimidopropanal was prepared by addition of
phthalimide to acrolein in the presence of tetrabutylammonium
hydroxide according to the procedure described by R. O. Atkinson
& F. Poppelsdorf, J. Chem. Soc. (1952) 2448. .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta.9.82 (t,1H,J=2); 7.85 (m,2H); 7.72
(m,2H); 4.04 (t,2H,J=7); 2.88 (dt, 2H,J=2,7). .sup.13C-NMR
(CDCl.sub.3, 100 MHz): .delta.199.36, 167.98, 134.10, 131.95,
123.36, 42.35, 31.67.
[0207] (b) The aldol adduct was prepared by reacting
3-phthalimidopropanal with N-propionyl-2-benzoxazolone according to
the procedure of paragraph C. .sup.13C-NMR (CDCl.sub.3, 100 MHz):
.delta.175.52, 168.75, 151.02, 142.17, 134.02, 132.05, 127.75,
125.46, 124.87, 123.35, 116.29, 109.85, 69.02, 44.00, 34.93, 33.11,
11.11.
[0208] HH.
(.+-.)-N-[(2R*,3S*)-(6-fluoro-2-methyl-3-hydroxyhexanoyl)]-2-be-
nzoxazolone 47
[0209] (a) 1-Bromo-3-fluoropropane is reacted with sodium cyanide
to give 4-fluorobutyronitrile. The nitrile is reduced with
diisobutylaluminum hydride to give 4-fluorobutyraldehyde.
[0210] (b) The aldol adduct is prepared by reacting 4-butyraldehyde
with N-propionyl-2-benzoxazolone according to the procedure of
paragraph C.
EXAMPLE 8
(.+-.)-N-[(2R*,3S*)-(3-cyclopropyl-2-methyl-3-hydroxypropionyl)]chlorzoxaz-
one
[0211] 48
[0212] A solution of N-propionylchlorzoxazone (2.25 g, 10 mmol) in
anhydrous CH.sub.2Cl.sub.2 (50 mL) was cooled to 3.degree. C. with
mechanical stirring under N.sub.2 atmosphere. TiCl.sub.4 (1.2 mL)
was added at a rate such that the internal temperature remained
below 10.degree. C. (ca. 1 minute). The resulting yellow slurry was
stirred vigorously for 5 minutes, then triethylamine (1.5 mL) was
added at a rate such that the internal temperature remained below
10.degree. C. (ca. 1 minutes). The resulting deep red solution was
stirred for 30 minutes. Cyclopropanecarboxaldehyde (0.75 mL) was
added in one portion. After stirring for 60 minutes, the reaction
was quenched by addition of 40 mL of 2 N HCl. The phases were
separated, and the aqueous phase was extracted once with 40-mL of
ether. The organic phases were combined, dried over MgSO.sub.4,
filtered, and concentrated under vacuum to a colorless oil.
Recrystallization from 1:1 ether/hexanes yielded 1.89 g of pure
product. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.8.12 (d, 1H, J=2
Hz); 7.25 (dd, 1 H, J=2, 8 Hz); 7.14 (d, 1H, J=8 Hz); 4.16 (dq, 1H,
J=4, 7 Hz); 3.28 (dd, 1H, J=4, 9 Hz); 2.22 (br s, 1H); 1.41 (d, 3H,
J=7 Hz); 1.10 (m, 1H); 0.56 (m, 2H); 0.37 (m, 2H). .sup.13C-NMR
(CDCl.sub.3, 100 MHz): .delta.175.24, 150.7, 140.62, 130.42,
128.54, 125.40, 116.73, 110.64, 44.45, 14.93, 11.03, 3.47,
2.70.
EXAMPLE 9
(.+-.)-N-[(2R*,3S*
)-(5-bromo-2-methyl-3-hydroxypentanoyl)]chlorzoxazone
[0213] 49
[0214] (a) A solution of 3-bromopropanal was prepared by bubbling
anhydrous HBr into an ice-cold solution of acrolein (5.6 g, 100
mmol) in dichloromethane (50 mL) containing 5 mg of
dicinnamylacetone as indicator. Once the solution stayed red for 5
minutes after cessation of HBr addition, the solution was checked
by .sup.1H-NMR by addition of 20 uL to 750 uL of CDCl.sub.3. NMR
revealed clean conversion to 3-bromopropanal, and relative
integration against the CH.sub.2Cl.sub.2 signal indicated a
concentration of 2.6 M 3-bromopropanal. Anhydrous MgSO.sub.4 was
added to the reaction mixture and stirred to absorb water. This
solution was filtered and used directly in the subsequent aldol
condensation. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.9.74 (t,
1H, J=1 Hz); 3.61 (t, 2H, J=7 Hz); 3.07 (dt, 2H, J=1,7 Hz).
.sup.13C-NMR (CDCl.sub.3, 400 MHz): .delta.198.95, 45.96,
23.35.
[0215] (b) A solution of N-propionylchlorzoxazone (11.3 g, 50 mmol)
in anhydrous CH.sub.2Cl.sub.2 (250 mL) was cooled to 3.degree. C.
with mechanical stirring under N.sub.2 atmosphere. TiCl.sub.4 (6.0
mL) was added at a rate such that the internal temperature remained
below 10.degree. C. (ca. 1 minute). The resulting yellow slurry was
stirred vigorously for 30 minutes, then triethylamine (7.5 mL) was
added at a rate such that the internal temperature remained below
10.degree. C. (ca. 1 minutes). The resulting deep red solution was
stirred for 30 minutes. The solution of 3-bromopropanal (25 mL, 60
mmol) was added in one portion. After stirring for 30 minutes, the
reaction was quenched by addition of 200 mL of 2 N HCl. The phases
were separated, and the aqueous phase was extracted once with
200-mL of ether. The organic phases were combined and filtered
through a pad of silica, washing the silica with ether. The
filtrate was evaporated to yield a tan solid, which was
recrystallized from ether by addition of hexane to yield 9.5 g
(52%) of the product as a colorless solid. .sup.1H-NMR (CDCl.sub.3,
400 MHz): .delta.8.14 (d, 1H, J=2 Hz); 7.28 (dd, 1 H, J=2, 8 Hz);
7.18 (d, 1H, J=8 Hz); 4.33 (dt, 1H, J=3,10 Hz); 3.96 (dq, 1H, J=4,
7 Hz); 3.61 (m, 2H); 2.3 (br s, 1H); 2.16 (m, 1H); 1.98 (m, 1H);
1.35 (d, 3H, J=7 Hz); .sup.13C-NMR (CDCl.sub.3, 100 MHz):
.delta.175.76, 150.74, 140.62, 130.54, 128.29, 125.64, 116.78,
110.78, 69.39, 43.71, 36.49, 30.14, 10.66.
EXAMPLE 10
(.+-.)-N-[(2R*,3S*)-(5-chloro-2-methyl-3-hydroxypentanoyl)]chlorzoxazone
[0216] 50
[0217] This was prepared according to the procedure for the
corresponding bromide of Example 9, using a solution of
3-chloropropanal in dichloromethane. .sup.1H-NMR (CDCl.sub.3, 400
MHz): .delta.8.12 (d, 1H, J=2 Hz); 7.26 (dd, 1 H, J=2, 8 Hz); 7.17
(d, 1H, J=8 Hz); 4.33 (m, 1H); 3.94 (dq, 1H, J 4, 7 Hz); 3.74 (m,
2H); 2.70 (br s, 1H); 2.05 (m, 1H); 1.90 (m, 1H); 1.34 (d, 3H, J=7
Hz). .sup.13C-NMR (CDCl.sub.3, 100 MHz): .delta.175.79, 150.74,
140.62, 130.53, 128.29, 125.63, 116.77, 110.78, 68.40, 43.76,
41.64, 36.37, 10.59.
EXAMPLE 11
(.+-.)-N-[(2R*,3S*)-3-hydroxy-2-vinyl-6-heptenoyl)]-2-benzoxazolone
[0218] 51
[0219] (a) N-crotonyl-2-benzoxazolone: A solution of
2-benzoxazolone (8.1 gm) in 60 mL of acetone was stirred with 8.3
gm of potassium carbonate while trans-crotonyl chloride (5.75 mL)
was added dropwise. After 16 hours, the mixture was poured into 150
mL of water, and the resulting precipitate was collected by vacuum
filtration and air dried. Recrystallization from ether/hexanes gave
10.5 gm (86%). .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.8.12
(m,1H); 7.40 (m,2H); 7.25 (m,4H); 2.07 (m,3H).
[0220] (b) Anhydrous CH.sub.2Cl.sub.2 (200 mL) was added to a flask
containing N-crotonyl-2-benzoxazolone (8.00 g, 39.4 mmol, 1.00 eq)
to make a 0.2 M solution which was cooled to -78.degree. C. in a
dry ice/acetone bath. Titanium (IV) chloride (4.41 mL, 40.2 mmol,
1.02 equiv) was added dropwise. The yellow slurry was stirred
vigorously for 20 min. Freshly distilled triethylamine (6.58 mL,
47.2 mmol, 1.20 equiv) was added dropwise. The color changed from
red-orange to deep purple during the addition. The solution was
stirred for 1.5 h at -78.degree. C. and 1.5 h at 0.degree. C. The
reaction mixture was returned to -78 .degree. C.; and freshly
distilled 4-penten-1-al (bp 102-103.degree. C.; 4.96 mL, 47.2 mmol,
1.2 equiv) was added dropwise over 15 min. The solution was stirred
for 2 h at -78.degree. C. and 1.5 h at 0.degree. C. The color
changed from purple to brown over this time. The reaction was
quenched with 2 N HCl.sub.(aq) (1.5 eq). The mixture was poured
into a separatory funnel, and the layers were separated. The
organic phase was vacuum filtered through a pad of silica. The
silica was washed with 3 volumes of ether, and all of the filtrate
was concentrated. The crude material was chromatographed over
silica (85:15 hexanes: EtOAc) to give 8.05 g (71%) of a faintly
colored oil. .sup.13C-NMR (CDCl.sub.3, 400 MHz): .delta.173.25,
150.90, 142.13, 137.91, 130.42, 127.67, 125.61, 124.91, 121.96,
116.19, 115.16, 109.92, 71.01, 53.79, 33.31, 29.85.
EXAMPLE 12
[0221]
(4S)-N-[(1S,2R)-2-hydroxy-5-cyclohexenyl-1-carboxyl]-2-benzoxazolon-
e 52
[0222] A solution of
(4S)-N-[(2S,3R)-3-hydroxy-2-vinyl-6-heptenoyl)]-4-ben-
zyl-2-oxazolidinone (35 mg) and 8 mg of bis(tricyclohexylphosphine)
benzylideneruthenium dichloride (Grubbs' catalyst) in
dichloromethane (5 mL) was heated at reflux under inert atmosphere
for 1.5 h. Chromatography yielded the cyclic metathesized product.
.sup.13C-NMR(CDCl.sub.3, 100 MHz): .delta.173.10, 153.56, 135.09,
130.30, 19.42, 128.95, 127.39, 121.36, 67.10, 66.30, 55.34, 45.57,
37.84, 27.44, 22.39.
EXAMPLE 13
Conversion to Thioesters
[0223] A. (+)-(2R*,3S*)-2-methyl-3-hydroxyhexanoate
N-propionylcysteamine thioester 53
[0224] One molar equivalent of sodium methoxide (25% w/v in
methanol; ca. 150 mL) is added in a slow stream to a solution of
N,S-dipropionylcysteamine (173 g) in methanol (910 mL) under
N.sub.2. When half of the calculated volume has been added, the
reaction is monitored by TLC (1:1 ethyl acetate/hexanes), and
methoxide addition is continued until complete conversion of the
N,S-dispropionylcysteamine to N-propionylcysteamine.
[0225] The resulting solution of sodium N-propionylcysteamine
thiolate is cannulated into a flask containing solid
(.+-.)-N-[(2R*,3S*)-(2-methyl-3-- hydroxyhexanoyl)]-2-benzoxazolone
(240 g) under N.sub.2. After 15 minutes, the reaction is quenched
with solid oxalic acid dihydrate (80.4 g), filtered, and
concentrated to a yellow oil. The residue is dissolved in 2:1
hexanes/ethyl acetate and submitted to batch elution chromatography
on SiO.sub.2. The silica is washed with 2:1 hexanes/ethyl acetate
to remove 2-benzoxazolone, then with ethyl acetate/methanol (9:1)
to elute the product thioester. Evaporation of the
thioester-containing eluent yields 222 g of the thioester (98%
yield) as a yellow oil, which crystallizes on standing; mp
37-39.degree. C. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.5.8 (br
s, 1H); 3.93 (dt, 1H); 3.44 (m, 2H); 3.03 (dt, 2H); 2.69 (dq, 1H);
2.19 (q, 2H); 1.47 (m, 2H); 1.36 (m, 2H); 1.19 (d, 3H); 1.14 (t,
3H); 0.92 (t, 3H).
[0226] The following are prepared according to the method of
paragraph A of this example.
[0227] B. (.+-.)-(2R*,3S*)-2-methyl-3-hydroxyhexanoate
N-acetylcysteamine thioester: 54
[0228] Prepared according to the method of paragraph A, by reaction
of N,S-diacetylcysteamine and
(.+-.)-N-[(2R*,3S*)-(2-methyl-3-hydroxyhexanoy-
l)]-2-benzoxazolone. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.6.1
(br s, 1H); 3.93 (dt, 1H); 3.44 (m, 2H); 3.03 (dt, 2H); 2.72 (dq,
1H); 1.97 (s,3H); 1.51 (m, 2H); 1.37 (m, 2H); 1.23 (d,3H); 1.14 (t,
3H); 0.94 (t, 3H). .sup.13C-NMR (CDCl.sub.3, 100 MHz):
.delta.204.05, 170.52, 71.87, 53.40, 39.33, 36.31, 28.53, 23.14,
19.16, 13.91, 11.14.
[0229] C. (.+-.)-(2R*,3S*)-2-methyl-3-hydrox-4-pentenoate
N-propionylcysteamine thioester 55
[0230] Prepared according to the method of paragraph A, by reaction
of N,S-dipropionyl-cysteamine and
(.+-.)-N-[(2R*,3S*)-(2-methyl-3-hydroxy-4--
pentenoyl)]-2-benzoxazolone.
[0231] D. (.+-.)-(2R*,3S*)-5-chloro-2-methyl-3-hydroxypentanoate
N-acetylcysteamine thioester 56
[0232] Prepared according to the procedure of paragraph A from
N,S-diacetylcysteamine and
(.+-.)-N-[(2R*,3S*)-5-chloro-2-methyl-3-hydrox-
ypentanoyl]chlorzoxazone. .sup.13C-NMR (CDCl.sub.3, 100 MHz):
.delta.203.47, 170.76, 69.01, 53.43, 41.79, 39.13, 36.80, 28.75,
23.17, 11.52.
[0233] E. (.+-.)-(2R*,3S*)-5-bromo-2-methyl-3-hydroxypentanoate
N-acetylcysteamine thioester 57
[0234] Prepared according to the procedure of paragraph A from
N,S-diacetylcysteamine and
(.+-.)-N-[(2R*,3S*)-5-bromo-2-methyl-3-hydroxy-
pentanoyl]-chlorzoxazone. .sup.13C-NMR (CDCl.sub.3, 100 MHz):
.delta.203.32, 170.82, 70.05, 53.44, 39.11, 37.00, 30.41, 28.74,
23.18, 11.64.
[0235] F. (.+-.)-(2R*,3S*)-5-azido-2-methyl-3-hydroxypentanoate
N-acetylcysteamine thioester 58
[0236] Prepared according to the procedure of paragraph A from
N,S-diacetylcysteamine and
(.+-.)-N-[(2R*,3S*)-5-azido-2-methyl-3-hydroxy-
pentanoyl]-2-benzoxazolone. .sup.13C-NMR (CDCl.sub.3, 100 MHz):
.delta.203.41, 170.75, 69.43, 53.62, 48.46, 39.12, 33.22, 28.73,
23.13, 11.57.
[0237] G.
(.+-.)-(2R*,3S*)-4-(2-methoxyethoxy)-2-methyl-3-hydroxybutanoate
N-propionylcysteamine thioester 59
[0238] Prepared according to the procedure of paragraph A from
N,S-dipropionylcysteamine and
(.+-.)-(.+-.)-N-[(2R*,3S*)-(4-(2-methoxyeth-
oxy)-2-methyl-3-hydroxybutanoyl)]-2-benzoxazolone. .sup.13C-NMR
(CDCl.sub.3, 100 MHz): .delta.202.77, 174.05, 72.83, 71.89, 71.15,
70.66, 58.97, 50.93, 39.30, 29.58, 28.61, 12.73, 9.72.
[0239] H.
(.+-.)-(2R*,3S*)-5-(2-pyrmidinylthio)-2-methyl-3-hydroxypentanoa-
te N-propionylcysteamine thioester 60
[0240] Prepared according to the procedure of paragraph A from
N,S-dipropionylcysteamine and
(.+-.)-(.+-.)-N-[(2R*,3S*)-(5-(2-pyrimidiny-
lthio)-2-methyl-3-hydroxypentanoyl)]-2-benzoxazolone. .sup.13C-NMR
(CDCl.sub.3, 100 MHz): .delta.203.23, 174.10, 157.30, 116.57,
70.12, 53.62, 39.28, 34.56, 29.60, 28.61, 27.24, 14.17, 12.26,
9.74
[0241] I. (.+-.)-(2R*,3S*)-2-methyl-3-hydroxy-6-heptenoate
N-acetylcysteamine thioester 61
[0242] Prepared according to the procedure of paragraph A from
N,S-diacetylcysteamine and
(.+-.)-N-[(2R*,3S*)-(2-methyl-3-hydroxy-6-hept-
enoyl)]-2-benzoxazolone. .sup.1H-NMR (CDCl.sub.3, 400 MHz):
.delta.5.91 (br s,1H), 5.82 (m,1H), 5.06 (dq,1H), 4.99 (dq,1H),
3.94 (m,1H), 3.47 (m,2h), 3.03 (m,2H), 2.73 (dq,1H), 2.56 (br
d,1H), 2.25 (m,1H), 2.15 (m,1H), 1.97 (s,3H), 1.60 (m,1H), 1.51
(m,1H), 1.23 (d,3H). .sup.13C-NMR (CDCl.sub.3, 100 MHz):
.delta.203.96, 170.44, 137.93, 115.18, 71.55, 53.40, 39.32, 33.30,
30.20, 28.60, 23.18, 11.26.
[0243] J. (.+-.)-(2R*,3S*)-2-methyl-3-hydroxyhexanoate
N-butyrylcysteamine thioester 62
[0244] Prepared according to the method of paragraph A using
N,S-dibutyrylcysteamine. .sup.1H-NMR (CDCl.sub.3): .delta.5.85 (br
s,1H), 3.93 (m,1H), 3.45 (m,2H), 3.02 (m,2H), 2.70 (dq,1H,J=3,7),
2.13 (m,3H), 1.65 (m,2H), 1.49 (m,2H), 1.33 (m,2H), 1.21
(d,3H,J,=7), 0.95 (t,3H,J=7), 0.92 (t,3H,J=7).
[0245] K. (.+-.)-(2R*,3S*)-2-methyl-3-hydroxyhexanoate
N-pentanoylcysteamine thioester 63
[0246] Prepared according to the method of paragraph A using
N,S-dipentanoylcysteamine. .sup.1H-NMR (CDCl.sub.3): .delta.5.81
(br s,1H), 3.92 (m, 1H); 3.44 (m,2H), 3.03 (m,2H), 2.70
(dq,1H,J=3,7), 2.15 (m,3H), 1.6 (m,2H), 1.5 (m,2H), 1.35 (m,4H),
1.21 (d,3H,J=7), 0.93 (t,3H,J=7), 0.91 (t,3H,J=7).
[0247] L. (.+-.)-(2R*,3S*)-2-methyl-3-hydroxyhexanoate
N-hexanoylcysteamine thioester 64
[0248] Prepared according to the method of paragraph A using
N,S-dihexanoylcysteamine. .sup.1H-NMR (CDCl.sub.3): .delta.5.83 (br
s,1H), 3.92 (m,1H); 3.44 (m,2H), 3.03 (m,2H), 2.69 (dq,1H,J=3,7),
2.14 (m,3H), 1.6 (m,2H), 1.45 (m,2H), 1.30 (m,6H), 1.20 (d,3H,J=7),
0.93 (t,3H,J=7), 0.88 (t,3H,J=7).
[0249] M. (.+-.)-(2R*,3S*)-2-methyl-3-hydroxyhexanoate
N-heptanoylcysteamine thioester 65
[0250] Prepared according to the method of paragraph A using
N,S-diheptanoylcysteamine. .sup.1H-NMR (CDCl.sub.3): .delta.5.83
(br s,1H), 3.92 (m,1H); 3.44 (m,2H), 3.03 (m,2H), 2.70
(dq,1H,J=3,7), 2.16 (m,3H), 1.6 (m,2H), 1.49 (m,2), 1.30 (m,8H),
1.20 (d,3H,J=7), 0.93 (t,3H,J=7), 0.87 (t,3H,J=7).
[0251] N. (.+-.)-(2R*,3S*)-2-methyl-3-hydroxyhexanoate
N-octanoylcysteamine thioester 66
[0252] Prepared according to the method of paragraph A using
N,S-dioctanoylcysteamine. .sup.1H-NMR (CDCl.sub.3): .delta.5.79 (br
s,1H), 3.93 (m,1H); 3.44 (m,2H), 3.03 (m,2H), 2.69 (dq,1H,J=3,7),
2.15 (m,3H), 1.6 (m,2H), 1.49 (m,2H), 130 (m,10H), 1.21 (d,3H,J=7),
0.93 (t,3H,J=7), 0.87 (t,3H,J=7).
EXAMPLE 14
(.+-.) -(2S*,3R*)-2-vinyl-3-hydroxy-6-heptenoate
N-propionylcysteamine thioester
[0253] 67
[0254] N,S-Dipropionyl cysteamine (4.28 g, 22.6 mmol, 1.00 eq) was
dissolved in methanol (36 mL). A 25 wt % solution of sodium
methoxide in methanol (3.89 mL, 17.0 mmol, 0.750 eq) was added
dropwise. The solution was stirred for 15 min and then cooled to
-78.degree. C. A methanolic solution of the aldol adduct (6.50 g,
22.6 mmol, 1.00 eq in 9 mL of MeOH) was added dropwise. The
reaction was stirred for 10 minutes at -78.degree. C. and brought
up to room temperature before quenching with solid oxalic acid
(1.42 g). Volatiles were removed in vacuo. The residue was
redissolved in ethyl acetate and washed with saturated NaHCO.sub.3
followed by saturated CuSO.sub.4. The organic layer was dried over
MgSO.sub.4, filtered, concentrated, and chromatographed on silica
gel (1:1 hexanes: EtOAc) to give 5.61 g (87.0%) of a colorless oil.
.sup.13C-NMR (CDCl.sub.3, 100 MHz): .delta.200.97, 174.15, 137.88,
131.18, 121.63, 115.06, 71.02, 64.45, 38.97, 33.38, 29.83, 29.54,
28.83, 9.68.
EXAMPLE 15
Production of 6-deoxyerythronolides
[0255] A. 15-methyl-6-deoxyerythronolide B 68
[0256] A seed culture of Streptomyces coelicolor K39-02/pJRJ2 was
made by inoculating 1 mL of frozen mycelium into a 2.8 L baffled
flask containing 500 mL of R2YE and shaking at 150-200
rpm/28-30.degree. C. for 2 days. A, 10 L stirred tank bioreactor
was prepared, filled with 10 L of FKA medium, autoclaved at
121.degree. C. for 30 min., allowed to cool, and then inoculated
with 400-500 mL of seed culture.
[0257] Temperature was maintained at 28-30.degree. C. with
agitation provided by 3 rushton impellers at 500-750 rpm, aeration
at .about.1 L/min., and pH controlled at 7.00 via automatic
addition of 1 N NaOH or 1 N H.sub.2SO.sub.4. Glucose consumption,
dissolved oxygen, pH, and cell mass were monitored. When the
glucose concentration dropped below 0.1 g/L, the culture was
supplemented with 10 g of (.+-.)-(2R*,3S*)-2-methyl--
3-hydroxyhexanoate N-propionylcysteamine thioester in 50 mL of
DMSO. Controlled feeding of glucose maintained a glucose
concentration of .about.0.5 g/L. Titers of
15-methyl-6-deoxy-erythronolide B were monitored by HPLC/MS, and
the culture was harvested by centrifugation when the maximum titer
was reached.
[0258] The 15-methyl-6-deoxyerythronolide B was purified by solid
phase extraction. Fermentation broth was cooled to 4-15.degree. C.,
and methanol was added to 10% (v/v). The broth was clarified by
centrifugation and loaded onto an XAD-16 resin (Rohm and Haas)
column (1 kg XAD/1 g 15-methyl-6-deoxyerythronolide B) at a flow
rate of 2-4 mL/cm.sup.2-min. The loaded resin was washed with 2
column volumes of 15% (v/v) methanol in water and the
15-methyl-6-deoxyerythronolide B was eluted from the resin with
acetone and collected in 1/2 column volume fractions. The fractions
containing 15-methyl-6-deoxyerythronolide B were identified by
thin-layer chromatography (ethyl acetate:hexanes 1:1) and HPLC/MS.
The acetone fractions containing 15-methyl-6-deoxyerythronolide B
were pooled, and the volatiles were removed under reduced pressure.
The resulting aqueous mixture is extracted with ethyl acetate. The
ethyl acetate extract was washed with saturated NaH.sub.2CO.sub.3
and brine solutions, dried over sodium or magnesium sulfate,
filtered, and concentrated to dryness under reduced pressure. The
crude material was purified by chromatography on silica gel using a
gradient of hexanes and ethyl acetate. Fractions containing the
product were pooled and concentrated to a pale yellow oil that
spontaneously crystallized. Recrystallization from ether-hexane
gave pure 15-methyl-6-deoxyerythronol- ide B. Mass spectrometry
shows [M+H]=401. .sup.13C-NMR (CDCl.sub.3, 100 MHz): .delta.213.57,
178.31, 79.51, 76.44, 74.47, 70.90, 43.95, 43.44, 40.88, 39.30,
37.66, 37.48, 35.52, 34.37, 19.45, 16.58, 14.68, 13.73, 13.23,
9.22, 6.87, 6.22.
[0259] B. 15-fluoro-6-deoxyerythronolide B 69
[0260] Prepared by feeding
(2S,3R)-5-fluoro-3-hydroxy-2-methylpentanoate N-acetyl-cysteamine
thioester to S. coelicolor CH999/pJRJ2 according to the method of
paragraph A. The crude material was purified by silica gel
chromatography using ethyl acetate/hexanes. APCI-MS: [M+H]=405.
.sup.19F-NMR (CDCl.sub.3, 376 MHz): .delta.-222.0 (relative to
CF.sub.3CO.sub.2H at .delta.-77.0). .sup.1H-NMR (CDCl.sub.3, 400
MHz): .delta.5.49 (m,1H); 4.94 (m,2H); 3.99 (m,1H); 3.90
(d,1H,J=10); 3.84 (d,1H,J=4); 3.70 (m,1H); 3.18 (br s,1H); 2.79
(m,1H); 2.77 (m,1H); 2.61 (m,1H); 2.47 (br s,1H); 2.20 (m,1H); 2.00
(m,1H); 1.92 (m,1H); 1.85 (m,1H); 1.70 (m,1H); 1.65 (dd,1H,J=4,10);
1.29 (d,3H,J=7); 1.24 (dd,1H,J=4,10); 1.07 (d,3H,J=7); 1.06
(d,3H,J=7); 1.05 (d,3H,J=7); 201 (d,3H,J=7);0.93 (d,3H,J=7).
.sup.13C-NMR (CDCl.sub.3, 100 MHz): .delta.213.70, 177.98, 80.68
(d,J.sub.CF=167 Hz), 79.34, 76.37, 70.84 (d,J.sub.CF=4 Hz), 70.74,
43.88, 43.27, 41.13, 39.54, 37.63, 37.52, 35.52, 33.34
(d,J.sub.CF=20 Hz), 16.63, 14.60, 13.32, 9.20, 6.92, 6.28.
[0261] C. 14,15-dehydro -6-deoxyerythronolide B 70
[0262] Prepared by feeding (2S,3R)-3-hydroxy-2-methyl-4-pentenoate
N-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according
to the method of paragraph A. The crude material was purified by
silica gel chromatography using ethyl acetate/hexanes. APCI-MS:
[M+H]=385. .sup.13C-NMR (CDCl.sub.3, 100 MHz): .delta.213.67,
177.51, 134.80, 116.58, 79.40, 76.47, 74.11, 70.84, 43.80, 43.16,
41.48, 39.58, 37.61, 37.42, 35.56, 16.60, 14.55, 13.34, 9.20, 6.91,
6.30.0
[0263] D. 15-chloro-6-deoxyerythronolide B 71
[0264] Prepared by feeding
(.+-.)-(2S*,3R*)-5-chloro-3-hydroxy-2-methylpen- tanoate
N-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according
to the method of paragraph A. The crude material is purified by
silica gel chromatography using ethyl acetate/hexanes. APCI-MS:
[M+H]=421.
[0265] E. 15-bromo-6-deoxyerythronolide B 72
[0266] Prepared by feeding
(.+-.)-(2S*,3R*)-5-bromo-3-hydroxy-2-methylpent- anoate
N-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according
to the method of paragraph A. The crude material was purified by
silica gel chromatography using ethyl acetate/hexanes. APCI-MS:
[M+H]=465, 467.
[0267] F. 15-dimethyl-6-deoxyerythronolide B 73
[0268] Prepared by feeding
(.+-.)-(2S*,3R*)-2,5-dimethyl-3-hydroxyhexanoat- e
N-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according
to the method of paragraph A. The crude material was purified by
silica gel chromatography using ethyl acetate/hexanes. APCI-MS:
[M+H]=415. .sup.13C-NMR (CDCl.sub.3, 100 MHz): .delta.213.98,
178.35, 79.58, 76.41, 72.87, 71.01, 43.96, 43.48, 41.26, 41.16,
39.35, 37.65, 37.43, 35.43, 25.33, 22.99, 21.95, 16.58, 14.56,
13.21, 9.29, 6.90, 6.24.
[0269] G. 15-phenyl-6-deoxyerythronolide B 74
[0270] Prepared by feeding
(.+-.)-(2S*,3R*)-5-phenyl-3-hydroxy-2-methylpen- tanoate
N-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according
to the method of paragraph A. The crude material was purified by
silica gel chromatography using ethyl acetate/hexanes. APCI-MS:
[M+H]=463. .sup.13C-NMR (CDCl.sub.3, 100 MHz): .delta.213.85,
178.30, 140.78, 128.55, 128.30, 126.23, 79.45, 76.37, 74.19, 70.90,
43.93, 43.37, 42.35, 41.04, 40.80, 39.47, 37.56, 37.56, 35.47,
34.41, 32.58, 16.65, 14.80, 13.28, 9.28, 6.95, 6.26.
[0271] H. 15-ethyl-6-deoxyerythronolide B 75
[0272] Prepared by feeding
(.+-.)-(2S*,3R*)-3-hydroxy-2-methylheptanoate N-acetylcysteamine
thioester to S. coelicolor CH999/pJRJ2 according to the method of
paragraph A. The crude material was purified by silica gel
chromatography using ethyl acetate/hexanes. APCI-MS: [M+H]=415.
.sup.13C-NMR (CDCl.sub.3, 100 MHz): .delta.213.65, 178.32, 79.51,
76.42, 74.74, 70.91, 43.95, 43.44, 40.84, 39.32, 37.65, 37.48,
35.50, 31.96, 28.37, 22.32, 16.58, 14.68, 13.93, 13.23, 9.20, 6.88,
6.23.
[0273] I. 15-propyl-6-deoxyerythronolide B 76
[0274] Prepared by feeding
(.+-.)-(2S*,3R*)-3-hydroxy-2-methyloctanoate N-acetylcysteamine
thioester to S. coelicolor CH999/pJRJ2 according to the method of
paragraph A. The crude material was purified by silica gel
chromatography using ethyl acetate/hexanes. APCI-MS: [M+H]=429.
.sup.13C-NMR (CDCl.sub.3, 100 MHz): .delta.213.66, 178.33, 79.51,
76.41, 74.76, 70.91, 43.95, 43.44, 40.85, 39.31, 37.65, 37.47,
35.50, 32.23, 31.38, 25.86, 22.48, 16.58, 14.68, 13.91, 13.22,
9.20, 6.88, 6.22.
[0275] J. 15-ethenyl-6-deoxyerythronolide B 77
[0276] Prepared by feeding
(.+-.)-(2S*,3R*)-3-hydroxy-2-methyl-6-heptenoat- e
N-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according
to the method of paragraph A. The crude material was purified by
silica gel chromatography using ethyl acetate/hexanes. APCI-MS:
[M+H]=413.
[0277] K. 13-desesthyl-13-phenyl-6-deoxyerythronolide B
[0278] Prepared by feeding
(.+-.)-(2S*,3R*)-3-phenyl-3-hydroxy-2-methylpro- panoate
N-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according
to the method of paragraph A. The crude material is purified by
silica gel chromatography using ethyl acetate/hexanes. APCI-MS:
[M+H]=435.
[0279] L. 12-ethenyl-12-desmethyl-6-deoxyerythronolide B 78
[0280] Prepared by feeding (2S,3R)-3-hydroxy-2-vinylpentanoate
N-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according
to the method of paragraph A. APCI-MS: [M+H]=398.
[0281] M. 12,15-bisethenyl-12-desmethyl-6-deoxyerythronolide B
79
[0282] Prepared by feeding (2S,3R)-3-hydroxy-2-vinyl-6-heptenoate
N-acetylcysteamine thioester to S. coelicolor CH999/pJRJ2 according
to the method of paragraph A. APCI-MS: [M+H]=425.
[0283] N. 15-azido-6-deoxyerythronolide B 80
[0284] Prepared according to the method of Example Y using
(2S*,3R*)-5-azido-2-methylpentanoate N-acetylcysteamine thioester.
APCI-MS: [MH.sup.+]=429.
EXAMPLE 16
Derivatives of dEB
[0285] A.
12-desmethyl-13-desethyl-12,13-(cyclohexenyl)-6-deoxyerythronoli-
de B 81
[0286] Prepared by treatment of
12,15-bisethenyl-12-desmethyl-6-deoxyeryth- ronolide B with Grubbs'
catalyst according to the procedure of Example 12.
[0287] B. 15-bromo-14-hydroxy-6-deoxyerythronolide B 82
[0288] A solution of 14,15-dehydro-6-deoxyerythronolide B in
aqueous acetonitrile is treated with N-bromosuccinimide. The
mixture is evaporated to dryness, and the product is isolated by
silica gel chromatography. .sup.13C-NMR (CDCl.sub.3, 100 MHz):
.delta.214.19, 175.82, 88.69, 81.52, 78.96, 77.46, 76.22, 49.43,
46.69, 43.76, 43.30, 38.50, 35.60, 34.30, 27.65, 18.06, 16.35,
15.53, 13.84, 13.06, 7.53.
EXAMPLE 17
Conversion of 6-deoxyerythronolides into ervthromycins
[0289] A. Fermentations were conducted in 10 L (and 150 L)
bioreactors. A 1 mL aliquot of frozen Sac. erythraea K40-67
mycelium was used to inoculate a seed culture in 500 mL of R2YE
medium. The culture was shaken at 150-200 rpm/28-30.degree. C. in a
2.8 L baffled Fembach flask for .about.48 hr. A 10 L stirred tank
bioreactor was prepared, filled with 10 L of R2YE medium (70 L for
the 150 L fermentation), autoclaved at 121.degree. C. for 45 min.,
allowed to cool, and then inoculated with 200 mL (1.4 L for the 150
L fermentation) of seed culture. Temperature was maintained at
28-30.degree. C. with agitation provided by 2 rushton impellers at
500-700 rpm, aeration at .about.1 L/min., and pH controlled at 7.20
via automatic addition of 1 N NaOH or 1 N H.sub.2SO.sub.4. Foam was
suppressed by addition of antifoam at 1 mL/L. The pH was controlled
to avoid potential product degradation into enol ether and
spiroketal. Sucrose consumption, glucose evolution, dissolved
oxygen, pH, and absorbance at 600 nm (cell mass) were monitored.
After 24-36 hr., the culture was fed 300 mg (1.62 g for the 150 L
fermentation) of a 6-dEB derivative compound dissolved in 3 mL (15
mL for the 150 L fermentation) of 100% ethanol. Fermentation
continued for .about.68-85 additional hr., and the fermentation
broth was harvested by centrifugation. Titers of erythromycin A, B,
C, and D analogs during the course of the fermentation were
determined by electrospray MS analysis.
[0290] The erythromycins produced were purified by solid phase
extraction. Fermentation broth was brought to pH 8.0 by addition of
NaOH and chilled to 4-15.degree. C., and ethanol was added (0.1 L/L
broth). The broth was clarified by centrifugation and loaded onto
an XAD-16 resin (Rohm and Haas) column (1 kg XAD/1 g erythromycin
derivative) at a flow rate of 2-4 mL/cm.sup.2-min. The loaded resin
was washed with 2 column volumes of 15% (v/v) ethanol in water and
the erythromycin derivative was eluted from the resin with acetone
and collected in 1/2 column volume fractions. The fractions
containing the erythromycin derivative were identified by
thin-layer chromatography and HPLC/MS.
[0291] The acetone fractions containing erythromycin analogs are
pooled and the volatiles are removed under reduced pressure. The
resulting aqueous mixture is extracted with ethyl acetate. The
ethyl acetate extract is washed with saturated NaHCO.sub.3 and
brine solutions, dried over sodium or magnesium sulfate, filtered,
and concentrated to dryness under reduced pressure. The crude
material is dissolved in dichloromethane and loaded onto a pad of
silica gel and washed with dichloromethane:methanol (96:4 v/v)
until the eluent is no longer yellow. The desired material is then
eluted with dichloromethane:methanol:triethy- lamine (94:4:2 v/v)
and collected in fractions. Fractions containing erythromycin are
identified by thin-layer chromatography, collected and concentrated
under reduced pressure. This material is recrystallized from
dichloromethane/hexanes.
[0292] B. 15-fluoroerythromycin A 83
[0293] Prepared by feeding 15-fluoro-6-deoxyerythronolide B to Sac.
erythraea according to the method of paragraph A. The crude
material was purified by silica gel chromatography. APCI-MS:
[M+H]=752. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.5.27 (1H, dd,
10, 2 Hz); 4.87 (1H, d, 5); 4.53 (2H, dtd, 40, 6, <1); 4.41 (1H,
d, 7); 3.98 (3H, m) 3.86 (1H, d, 1); 3.56 (1H, d, 7); 3.48 (1H, m);
3.31 (3H, s); 3.23, (1H, dd, 10, 7); 3.19 (1H, br s); 3.08 (1H, qd,
7, 1); 3.00 (1H, br s, 8); 2.84 (1H, qd, 7.1); 2.70 (1H, m); 2.46
(1H, 7,4); 2.36 (1H, d, 16), 2.30 (6H, s); 2.03-1.95 (2H, m); 1.94
(1H, m) 1.73 (1H,br d, 15); 1.69 (1H, br d, 14); 1.57 (1H, m); 1.47
(3H, s); 1.28 (3H, d, 6); 1.24 (3H, s);1.22 (3H, d, 6); 1.21 (1H,
ovrlp); 1.17 (3H, d, 7); 1.15 (3H, d, ovrlp); 1.14 (3H, d, ovrlp);
1.10 (3H, d, 7). .sup.13C NMR (100 MHz, CDCl.sub.3): .delta.222.0,
175.4, 103.2, 96.3, 83.4, 82.3 (d, 170 Hz), 79.8, 77.9, 75.1, 74,3,
72.6, 72.5 (d, 4 Hz), 70.9, 68.9, 68.5, 65.6, 65.6, 49.5, 45,2,
44.8, 40.3, 39.6, 38.5, 37.7, 34.9, 29.5, 29.4 (d, 20 Hz), 28.7,
27.0, 21.5, 21.4, 18.6, 18.2, 16.1 15,3, 11.9, 9.1.
[0294] C. 15-ethenylerythromycin A 84
[0295] Is prepared by feeding 15-ethenyl-6-deoxyerythronolide B to
Sac. erythraea according to the method of paragraph A. The crude
material is purified by silica gel chromatography.
EXAMPLE 18
15-(2-(3-guinolyl)ethyl)erythromycin A
[0296] 85
[0297] (1) A solution of 15-ethenylerythromycin A (1 mmol) in 5 mL
of dichloromethane is treated with benzoic anhydride (1.5 mmol) and
triethylamine (1.5 mmol) at ambient temperature for 30 hours.
Aqueous 5% Na.sub.2CO.sub.3 is added and stirred for 30 minutes,
then the mixture is extracted with dichloromethane. The organic
extracts are combined, washed with saturated aqueous NaHCO.sub.3
followed by brine, dried over MgSO.sub.4, filtered, and evaporated.
Chromatography on silica gel provides pure
2'-O-benzoyl-15-ethenylerythromycin A.
[0298] (2) A mixture of 2'-O-benzoyl-15-ethenylerythromycin A (1
mmol), palladium diacetate (0.2 mmol), tritolylphosphine (0.4
mmol), and 3-bromoquinoline (2 mmol) in 8 mL of deoxygenated
acetonitrile is cooled to -78.degree. C., degassed, and sealed in a
reaction tube. The mixture is kept at 50.degree. C. with stirring
for 30 hours, then cooled and opened and the acetonitrile removed
under vacuum. The residue is dissolved in ethyl acetate and washed
successively with 5% aqueous Na.sub.2CO.sub.3, 2% aqueous Tris, and
brine. After drying over Mg.sub.2SO.sub.4, the mixture is filtered
and evaporated. Silica gel chromatography gives pure
2'-O-benzoyl-15-(2-(3-quinolyl)ethyl)erythromyc- in A.
[0299] (3) A solution of
2'-O-benzoyl-15-(2-(3-quinolyl)ethyl)erythromycin A (1 mmol) in
methanol (10 mL) is heated at reflux for 6 hours, then evaporated.
The residue is purified by silica gel chromatography to yield
15-(2-(3-quinolyl)ethyl)erythromycin A.
EXAMPLE 19
Preparation of Polystyrene-Supported 2-Benzimidazolone
[0300] (I) A mixture of 2-hydroxybenzimidazole,
6-(acetylthio)-1-bromohexa- ne, and triethylamine in acetonitrile
is heated at reflux to prepare
1-(6-(acetylthio)hexyl)-2-benzimidazolone.
[0301] (2) A solution of 1-(6-(acetylthio)hexyl)-2-benzimidazolone
in methanol is treated with one equivalent of sodium methoxide to
prepare 1-(6-mercaptohexyl)-2-benzimidazolone.
[0302] (3) Merrifield resin (chloromethylated
polystyrene-divinylbenzene) is suspended in dichloromethane by
gentle stirring, and treated with
1-(6-mercaptohexyl)-2-benzimidazolone and triethylamine to prepare
polystyrene-supported 2-benzimidazolone.
EXAMPLE 20
Preparation of Polystyrene-Supported
(4S)-4-benzyl-2-imidazolidinone
[0303] (1) N-ethoxycarbonyl-(L)-phenylalinal is prepared from
commercially-available N-ethoxycarbonyl-(L)-phenylalanine according
to the method described for N-tbutoxycarbonyl-(L)-leucinal by O. P.
Goel, et al., Organic Syntheses (1988) 67:69. This aldehyde is
dissolved in methanol and treated with 1,4-diaminobutane, acetic
acid, and sodium cyanoborohydride at 0.degree. C. The resulting
amine is isolated by chromatography, then heated under vacuum with
removal of ethanol to provide
(4S)-1-(4-aminobutyl)-4-benzyl-2-imidazolinone.
[0304] (2) Carboxypolystyrene resin is suspended by gentle stirring
in dichloromethane and treated sequentially with
1-hydroxybenzotriazole and dicyclohexylcarbodiimide. After 30
minutes, (4S)-1-(4-aminobutyl)-4-benzy- l-2-imidazolinone is added.
The solution is checked periodically for disappearance of the
amine. The resin is collected by vacuum filtration, washed with
dichloromethane and dried.
EXAMPLE 21
General Solid-Phase Synthesis of
(2S,3R)-2-Methyl-3-hydroxy-diketide thioesters
[0305] (1) Polystyrene-supported (4S)-4-benzyl-2-imidazolidinone is
suspended in tetrahydrofuran and treated with excess propionic
anhydride, triethylamine, and catalytic 4-dimethylaminopyridine
overnight. The resin is collected by vacuum filtration and washed
with water followed by acetone, then dried under vacuum to yield
propionylated resin.
[0306] (2) The propionylated resin is suspended by shaking in
anhydrous dichloromethane in a bottom-fritted reaction vessel under
inert atmosphere and cooled to 0.degree. C. A small molar excess of
dibutylboron triflate is added and the vessel contents are shaken
for 30 minutes. A small molar excess of triethylamine is added and
the vessel contents are shaken for another 30 minutes. The liquid
phase is drained from the vessel through the bottom frit using gas
pressure, and is replaced with clean dichloromethane containing a
small molar excess of the aldehyde component. After shaking for 4
hours, the solvent is drained from the vessel via the frit and the
resin is washed with clean dichloromethane. The resin is suspended
in a mixture of phosphate buffer, pH 7, methanol, and
H.sub.2O.sub.2 and shaken for 1 hour at 0.degree. C. The solution
is drained and the resin is washed sequentially with water,
saturated NaHCO.sub.3, water, methanol, and tetrahydrofuran, then
dried under vacuum.
[0307] (3) An N-acylcystearnine is dissolved in tetrahydrofuran
under inert atmosphere and cooled to -78.degree. C. One molar
equivalent of n-butyllithium is added, resulting in a white
suspension. Addition of one molar equivalent of trimethylaluminum
results in a clear solution of the aluminate salt. The resulting
solution is added to the diketide-containing resin, and the mixture
is shaken to release the diketide thioester. The solution is
neutralized with oxalic acid, collected from the resin by vacuum
filtration via the frit, and evaporated to dryness. The residue is
resuspended in ethyl acetate and washed with saturated aqueous
CuSO.sub.4 followed by brine. After drying over MgSO.sub.4, the
solution is filtered and evaporated. Chromatography yields the
purified diketide thioester.
EXAMPLE 22
Preparation of Polystyrene-Supported 2-Benzoxazolone
[0308] (1) A mixture of chlorzoxazone,
3-(t-butoxycarbonylamino)-1-propene- , palladium diacetate,
tritolylphosphine, and acetonitrile is cooled to -78.degree. C.,
degassed, and sealed in a reaction tube. The mixture is kept at
50.degree. C. with stirring for 60 hours, then cooled and opened
and the acetonitrile removed under vacuum. The residue is dissolved
in ethyl acetate and washed successively with 5% aqueous NaHCO3 and
brine. After drying over Mg2SO4, the mixture is filtered and
evaporated. Silica gel chromatography gives
5-(3-(t-butoxycarbonylamino)-1-propenyl)-2-benzo- xazolone.
[0309] (2) A solution of
5-(3-(t-butoxycarbonylamino)-1-propenyl)-2-benzox- azolone in
trifluoroacetic acid is stirred at ambient temperature for 30 min,
then evaporated to dryness to yield
5-(3-amino-1-propenyl)-2-benzoxa- zolone.
[0310] (3) Carboxypolystyrene resin is suspended by gentle stirring
in dichloromethane and treated sequentially with
1-hydroxybenzotriazole and dicyclohexylcarbodiimide. After 30
minutes, 5-(3-amino-1-propenyl)-2-benz- oxazolone is added. The
solution is checked periodically for disappearance of the amine.
The resin is collected by vacuum filtration, washed with
dichloromethane and dried.
EXAMPLE 23
General Solid-Phase Synthesis of Racemic
2-Methyl-3-hydroxy-diketide thioesters
[0311] (1) Polystyrene-supported 2-benzoxazolone is suspended in
acetone and treated with excess propionic anhydride and
triethylamine overnight. The resin is collected by vacuum
filtration and washed with water followed by acetone, then dried
under vacuum to yield propionylated resin.
[0312] (2) The propionylated resin is suspended by shaking in
anhydrous dichloromethane in a bottom-fritted reaction vessel under
inert atmosphere and cooled to 0.degree. C. A small molar excess of
titanium tetrachloride is added and the vessel contents are shaken
for 30 minutes. A small molar excess of triethylamine is added and
the vessel contents are shaken for another 30 minutes. The liquid
phase is drained from the vessel through the bottom frit using gas
pressure, and is replaced with clean dichloromethane containing a
small molar excess of the aldehyde component. After shaking for 4
hours, the solvent is drained from the vessel via the frit and the
resin is washed with clean dichloromethane. The resin is washed
with 1 N HCl to remove titanium residues, followed by water and
methanol. This provides 2-methyl-3-hydroxy-diketides bound to
polystyrene.
[0313] (3) An N,S-diacylcysteamine is dissolved in methanol and
treated with one molar equivalent of methanolic sodium methoxide.
The resulting solution is added to the diketide-containing resin,
and the mixture is shaken to release the diketide thioester. The
solution is neutralized with oxalic acid, collected from the resin
by vacuum filtration via the frit, and evaporated to dryness. The
residue is resuspended in ethyl acetate and washed with saturated
aqueous CuSO.sub.4 followed by brine. After drying over MgSO.sub.4,
the solution is filtered and evaporated. Chromatography yields the
purified racemic diketide thioester.
EXAMPLE 24
15-(2-(3-quinolyl)ethyl)-3-descladinosyl-3-oxo-6-O-methylerythromycin
A 11,12-cyclic carbamate
[0314] 86
[0315] A. 15-(2-(3-guinolyl)ethyl)erythromycin A-9-oxime
[0316] 15-(2-(3-quinolyl)ethyl)erythromycin A (25.7 g, 28.9 mmol,
1.00 eq) is suspended in 42 mL of 2-propanol. Hydroxylamine (50 wt
% in H.sub.2O, 22.2 mL, 375 mmol, 13.0 eq) is added. The mixture is
stirred until homogeneous. Glacial HOAc is added. The solution is
stirred at 50.degree. C. for 11 h. Saturated NaHCO.sub.3 is added.
The mixture is concentrated and extracted with CHCl.sub.3
(4.times.400 mL); washed with NaHCO.sub.3 and water. The combined
aqueous layers are back-extracted with 400 mL CHCl.sub.3. The
combined organic phases are washed with brine, dried over
Na.sub.2SO.sub.4, filtered, and concentrated to yield the crude
material. This is carried on without further purification.
[0317] B. 15-(2-(3-guinolyl)ethyl)erythromycin
A-9-(isopropoxycyclohexyl)o- xime
[0318] The crude 15-(2-(3-quinolyl)ethyl)erythromycin A-9-oxime
from above is dissolved in 72 mL of anhydrous CH.sub.2Cl.sub.2, and
1,1-diisopropoxycyclohexane (29.2 mL, 140 mmol, 4.86 eq) is added
dropwise. A solution of pyridinium p-toluenesulfonate (10.5 g, 41.9
mmol, 1.45 eq) in CH.sub.2Cl.sub.2 (36 mL) is added dropwise.
Dichloromethane (200 mL) is added after 15 h. The solution is
washed with NaHCO.sub.3 (2.times.100 mL) and water (100 mL). The
combined aqueous phases are back-extracted with 100 mL
CH.sub.2Cl.sub.2. The combined organic layers are washed with
brine, dried over MgSO.sub.4, filtered, and concentrated. The
material is chromatographed over silica gel to give the desired
product.
[0319] C.
2',4"-Bis(O-trimethylsilyl)-15-(2-(3-quinolyl)ethyl)erythromycin
A-9-(isopropoxycyclohexyl)oxime.
[0320] The 15-(2-(3-quinolyl)ethyl)erythromycin
A-9-(isopropoxycyclohexyl)- oxime (22.2 g, 21.3 mmol, 1.0 eq) is
dissolved in 54 mL anhydrous CH.sub.2Cl.sub.2 and cooled in an
ice/water bath. A mixture of chlorotrimethylsilane (4.05 mL, 31.9
mmol, 1.5 eq), N-(trimethylsilyl)-imidazole (7.81 mL, 53.2 mmol,
2.5 eq), and CH.sub.2Cl.sub.2 (18 mL) is added dropwise. The
reaction is stirred for 15 minutes after complete addition and
quenched with 600 mL EtOAc. The mixture is washed with sat.
NaHCO.sub.3 (2.times.200 mL), water (200 mL), and brine (200 mL).
The organic layer is dried over MgSO.sub.4, filtered, and
concentrated to yield the crude product which was carried on
without further purification.
[0321] D.
2',4"-Bis(O-trimethylsilyl)-6-O-methyl-15-(2-(3-guinolyl)ethyl)e-
rythromycin A-9-(isopropoxycyclohexyl)oxime
[0322] Crude
2',4"-bis(O-trimethylsilyl)-15-(2-(3-quinolyl)ethyl)erythromy- cin
A-9-(isopropoxycyclohexyl)oxime is dissolved in anhydrous
tetrahydrofuran (41 mL) and cooled to 10.degree. C. Anhydrous
methylsulfoxide (41.4 mL) and methyl bromide (2.0 M in ether, 20.7
mL, 41.4 mmol, 2.0 eq) are added. A 1.0 M solution of potassium
t-butoxide in THF (41.4 mL, 41.4 mmol, 2.0 eq) is diluted wtih
anhydrous methylsulfoxide (41.4 mL). This is added to the reaction
mixture at a rate of 0.5 eq/hr. The reaction is monitored by TLC
(5:1 toluene:acetone). The reaction is quenched by the addition of
ethyl acetate (200 mL) and sat. NaHCO.sub.3 (70 mL). The mixture is
transferred to a separatory funnel and diluted with 850 mL of ethyl
acetate. The organic phase is washed with sat. NaHCO.sub.3, water,
and brine (300 mL each). The resulting emulsion is filtered through
Celite. The separated organic phase is then dried over MgSO.sub.4,
filtered, and concentrated to give the crude product which is
carried on without further purification.
[0323] E. 6-O-Methyl-15-(2-(3-quinolyl)ethyl)erythromycin
A-9-oxime
[0324] The crude
2',4"-bis(trimethylsilyl)-6-O-methyl-15-(2-(3-quinolyl)et-
hyl)erythromycin A-9-(isopropoxycyclohexyl)oxime from above is
dissolved in acetonitrile (110 mL). Glacial acetic acid (67 mL)
diluted with water (55 mL) is added slowly. The solution is stirred
8 h. Toluene and 2-propanol are added, and the solution is
concentrated. The product is then dissolved in toluene and
concentrated twice to give the crude product which was carried on
without further purification.
[0325] F. 6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycin A
[0326] The crude 6-O-methyl-15-(2-(3-quinolyl)ethyl)erythromycin
A-9-oxime from above and sodium hydrosulfite (23.1 g, 113 mmol,
5.63 eq) are placed in a round-bottom flask equipped with a
condenser and flushed with N.sub.2. Ethanol (140 mL) and water (140
mL) are added. Formic acid (3.75 mL, 95.4 mmol, 4.77 eq) is added
dropwise. The mixture is stirred at 80 C. for 4.5 h. After the
solution returned to room temperature, sat. NaHCO.sub.3 was added.
The pH is adjusted to 9-10 with 6 N NaOH. The mixture is then
extracted with 3.times.400 mL of ethyl acetate. The combined
organic phases are washed with sat. NaHCO.sub.3 then water (250 mL
each). The combined aqueous phases are back-extracted with ethyl
acetate (400 mL). The combined organic phases are washed with
brine, dried over MgSO.sub.4, filtered, and concentrated to give
the crude product which was carried on without further
purification. Pure product can be obtained by chromatography on
silica gel.
[0327] G.
6-O-Methyl-15-(2-(3-quinolyl)ethyl)-3-descladinosylerythromycin
A
[0328] The crude 6-O-methyl-i 5-(2-(3-quinolyl)ethyl)erythromycin A
is stirred in 280 mL of 0.5 M HCl for 3 h. The pH is adjusted to
9-10 with 6 N NaOH. The precipitate is collected by vacuum
filtration and washed with water. The mother liquor is extracted
with 3.times.400 mL ethyl acetate. The combined organic phases are
washed with sat. NaHCO.sub.3 and water. The combined aqueous phases
are back-extracted with ethyl acetate. The combined organic phases
are washed with brine, dried over MgSO.sub.4, filtered, and
concentrated. The combined product is chromatographed over silica
gel the desired product as a white solid.
[0329] H.
2'-O-Acetyl-6-O-methyl-15-(2-(3-guinolyl)ethyl)-3-descladinosyle-
rythromycin A
[0330] 6-O-Methyl-15-(2-(3-quinolyl)ethyl)-3-descladinosyl
erythromycin A (11.5 g, 15.5 mmol, 1.0 eq) is dissolved in 40 mL
ethyl acetate. A solution of acetic anhydride (2.92 mL, 31.0 mmol,
2.0 eq) in ethyl acetate (35 mL) is added dropwise. The reaction is
stirred for 30 min and then concentrated. The material is
chromatographed over silica gel to give the desired product as a
white solid.
[0331] I.
2'-O-Acetyl-3-descladinosyl-3-oxo-6-O-methyl-15-(2-(3-quinolyl)e-
thyl)erythromycin A
[0332]
2'-O-Acetyl-6-O-methyl-15-(2-(3-quinolyl)ethyl)-3-descladinosyl
erythromycin A (10 g, 12.8 mmol, 1.0 eq) and
1-[3-(dimethylamino)propyl]-- 3-ethylcarbodiimide hydrochloride
(16.51 g, 86.1 mmol, 6.7 eq) are combined in a round-bottom flask
and flushed with N.sub.2. The solids are dissolved in anhydrous
CH.sub.2Cl.sub.2 (64 mL) and cooled in an ice water bath. Anhydrous
DMSO (15.5 mL, 218 mmol, 17 eq) is added. A solution of pyridinium
trifluoroacetate (12.14 g, 62.9 mmol, 4.9 eq) in CH.sub.2Cl.sub.2
(47 mL) is added over 3 h. The solution is diluted with 600 mL of
ethyl acetate and washed with sat. NaHCO.sub.3, water, and brine
(200 mL each). The organic phase is dried over MgSO.sub.4,
filtered, and concentrated. Chromatography over silica gel gives
the desired product.
[0333] J.
2'-O-Acetyl-3-oxo-3-descladinosyl-11-methanesulfonyl-6-O-methyl--
15-(2-(3-quinolyl)ethyl)erythromycin A
[0334]
2'-O-Acetyl-3-descladinosyl-3-oxo-6-O-methyl-15-(2-(3-quinolyl)ethy-
l)erythromycin A is dissolved in freshly distilled pyridine (35 mL)
and cooled in an ice water bath. Methanesulfonyl chloride is added
dropwise. The reaction is allowed to come to ambient temperature
and stirred overnight. Ethyl acetate (700 mL) is added, and the
solution is washed with sat. NaHCO.sub.3, water, and brine (200 mL
each). The organic phase is dried over MgSO.sub.4, filtered, and
concentrated. Chromatography over silica gel gives the desired
compound.
[0335] K.
2'-O-Acetyl-10,11-anhydro-3-descladinosyl-3-oxo-6-O-methyl-15-(2-
-(3-quinolyl)ethyl)erythromycin A
[0336]
2'-O-Acetyl-3-oxo-3-descladinosyl-11-methanesulfonyl-6-O-methyl-15--
(2-(3-quinolyl)ethyl)erythromycin A (6 g, 6.98 mmol, 1.0 eq) is
dissolved in acetone (23 mL). 1,8-Diazabicyclo(5.4.0)undec-7-ene
(5.22 mL, 34.9 mmol, 5.0 eq) is added dropwise. The reaction is
stirred at ambient temperature for 4 h and then concentrated.
Chromatography over silica gel gave the desired compound.
[0337] L.
3-descladinosyl-3-oxo-6-O-methyl-15-(2-(3-quinolyl)ethyl)erythro-
mycin A 11,12-cyclic carbamate
[0338] A solution of
2'-O-Acetyl-10,11-anhydro-3-descladinosyl-3-oxo-6-O-m-
ethyl-15-(2-(3-quinolyl)ethyl)erythromycin A in dry tetrahydrofuran
is added to a stirred suspension of NaH (3 eq.) in THF cooled to
-10.degree. C. To this is added a solution of carbonyldiimidazole
(10 eq.) in THF/DMF (5:3), and the mixture is stirred for 2 hours.
The reaction is warmed to ambient temperature and diluted with
concentrated aqueous ammonia and stirred overnight. The mixture is
diluted with ethyl acetate and washed with aq. NaHCO.sub.3 and
brine, dried over MgSO.sub.4, and evaporated. Chromatography on
silica gel yields the product.
[0339] The following provides additional products of the
benzoxazolones.
[0340] A. Methyl (.+-.)-(2S*,3R*)-3-hydroxy-2-methythexanoate
87
[0341] 4-Dimethylaminopyridine (25 mg, 0.2 mmol) was added to a
solution of
(.+-.)-N-[(2S*,3R*)-(2-methyl-3-hydroxyhexanoyl)]-2-benzoxazolone
(263 mg, 1.0 mmol) in methanol (10 mL). The reaction mixture was
stirred overnight and the methanol was removed at reduced pressure.
The resulting oil was redissolved in ether (50 mL) and washed with
1 N sodium hydroxide (2.times.10 mL), 2 N HCl (10 mL), and brine
(10 mL), dried with magnesium sulfate and concentrated at reduced
pressure to give a clear oil (118 mg, 74%). .sup.1H-NMR
(CDCl.sub.3, 400 MHz) .delta.3.90 (m, 1 H),2.53 (dq, J=3,3 Hz 1 H),
2.45 (br s, 1 H), 1.49 (m, 2 H), 1.34 (m, 2 H), 1.76 (d, J=7 Hz),
0.93 (t, J=7 Hz). .sup.13C-NMR (CDCl.sub.3, 100 MHz) .delta.176.3,
71.4, 51.5, 44.3, 36.0, 19.0, 13.8, 10.6.
[0342] B. N-Benzyl (.+-.)-(2S*,3R*)-3-hydroxy-2-methylhexanamide
88
[0343] Benzylamine (0.6 mL, 5.5 mmol) is added dropwise to a
solution of
N-[(2S*,3R*)-3-hydroxy-2-methylhexanoyl]-2-benzoxazolinone (1.31 g,
5.0 mmol) in 10 mL of tetrahydrofuran. A mildly exothermic reaction
ensues. After 15 min, the solvent is evaporated. The residue is
redissolved in 50 mL of CH.sub.2Cl.sub.2 and washed successively
with equal volumes of 1 N HCl, 1 N NaOH, water, and brine. After
drying over MgSO4, the solution is evaporated to yield 1.12 g (95%
yield) of the product as a white solid which was recrystallized
from ethyl acetate/hexanes as white needles, mp 114-115.degree. C.
.sup.1H-NMR (d.sub.6-DMSO, 400 MHz) .delta.8.27 (t, J=6, 1 H), 7.30
(m, 2 H), 7.22 (m, 3 H), 4.49 (d,J=6, 1 H), 4.27 (dd,J=6,15, 1 H),
4.20 (dd,J=6,15, 1 H), 3.44 (m, 1 H), 2.20 (q,J=7,1 H), 1.43 (m, 1
H), 1.22 (m, 3 H), 1.04 (d,J=7,3 H), 0.79 (t,J=7,3 H). .sup.13C-NMR
(d.sub.6-DMSO, 100 MHz): 175.2, 140.2, 128.6, 127.6, 127.1, 71.8,
46.9, 42.2, 37.6, 19.0, 14.7, 14.5.
* * * * *