U.S. patent application number 12/141732 was filed with the patent office on 2009-04-30 for benzoquinone ansamycins.
This patent application is currently assigned to Kosan Biosciences, Inc.. Invention is credited to Li Feng, C. Richard Hutchinson, Robert Johnson, David C. Myles, Daniel SANTI, Zong-Qiang Tian, Yi-Qing Zhou.
Application Number | 20090111869 12/141732 |
Document ID | / |
Family ID | 27539665 |
Filed Date | 2009-04-30 |
United States Patent
Application |
20090111869 |
Kind Code |
A1 |
SANTI; Daniel ; et
al. |
April 30, 2009 |
BENZOQUINONE ANSAMYCINS
Abstract
The invention relates to benzoquinone ansamycin analogs useful
for the treatment of cancer and other diseases or conditions
characterized by undesired cellular proliferation or
hyperproliferation. Therapies involving the administration of such
benzoquinone ansamycin analogs, optionally in combination with an
inhibitor of an HSP90 client protein, are useful to treat cancer
and non-cancerous disease conditions.
Inventors: |
SANTI; Daniel; (San
Francisco, CA) ; Myles; David C.; (Kensington,
CA) ; Tian; Zong-Qiang; (Fremont, CA) ;
Hutchinson; C. Richard; (San Mateo, CA) ; Johnson;
Robert; (Lafayette, CA) ; Zhou; Yi-Qing;
(Lafayette, CA) ; Feng; Li; (Fremont, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
12531 HIGH BLUFF DRIVE, SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Assignee: |
Kosan Biosciences, Inc.
Hayward
CA
|
Family ID: |
27539665 |
Appl. No.: |
12/141732 |
Filed: |
June 18, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11056470 |
Feb 11, 2005 |
7405208 |
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12141732 |
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10212962 |
Aug 5, 2002 |
6872715 |
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11056470 |
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60310779 |
Aug 6, 2001 |
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60389255 |
Jun 14, 2002 |
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60393929 |
Jul 3, 2002 |
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60395275 |
Jul 12, 2002 |
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Current U.S.
Class: |
514/460 ;
514/183; 514/449 |
Current CPC
Class: |
C07D 491/08 20130101;
A61P 35/00 20180101; C07D 225/06 20130101 |
Class at
Publication: |
514/460 ;
514/183; 514/449 |
International
Class: |
A61K 31/395 20060101
A61K031/395; A61P 35/00 20060101 A61P035/00; A61K 31/337 20060101
A61K031/337; A61K 31/366 20060101 A61K031/366 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] The present invention was made in part under NIH Grant No.
R43 CA96262-01. The United States government has certain rights in
this invention.
Claims
1. A method for treatment of a disease or condition characterized
by undesired cellular proliferation or hyperproliferation in a
subject suffering therefrom, comprising the steps of: (a)
administering to said subject a substantially sub-toxic dose of an
Hsp90 client protein inhibitor, wherein the Hsp90 client protein
inhibitor is a microtubule stabilizing agent; (b) waiting a period
of time sufficient to allow development of a substantially
efficacious response; and (c) administering to said subject a
synergistic dose of a benzoquinone ansamycin.
2. The method of claim 1 wherein the benzoquinone ansamycin is
selected from the group consisting of 17-allylamino-17
desmethoxy-geldanamycin, and
17-(2-(dimethylamino)ethylamino-17-desmethoxygeldanamycin.
3. The method of claim 1, wherein the disease is breast cancer.
4. A method for treatment of a disease or condition characterized
by undesired cellular proliferation or hyperproliferation in a
subject suffering therefrom, comprising the steps of: (a)
administering to said subject a synergistic dose of a benzoquinone
ansamycin; (b) waiting a period of time sufficient to allow
development of a substantially efficacious response; and (c)
administering to said subject a sub-toxic dose of an Hsp90 client
protein inhibitor, wherein the Hsp90 client protein inhibitor is a
microtubule stabilizing agent.
5. The method of claim 4 wherein the microtubule stabilizing agent
is selected from the group consisting of paclitaxel, an epothilone,
discodermolide, and laulimalide.
6. The method of claim 4 wherein the benzoquinone ansamycin is
selected from the group consisting of 17-allylamino-17
desmethoxy-geldanamycin, and 17 (2
(dimethylamino)ethylamino-17-desmethoxygeldanamycin.
7. The method of claim 4, wherein the disease is breast cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 11/056,470, filed on Feb. 11, 2005, which is a Divisional
of U.S. Ser. No. 10/212,962 filed Aug. 5, 2002, issuing on Mar. 29,
2005 as U.S. Pat. No. 6,872,715, which claims priority to U.S.
Provisional Application 60/310,779, filed Aug. 6, 2001, entitled
"Geldanamycin Analogs," and to U.S. Provisional Applications
60/389,255, filed Jun. 14, 2002; 60/393,929, filed Jul. 3, 2002;
and 60/395,275, filed Jul. 12, 2002; each entitled "Recombinant
Geldanamycin Polyketide Synthase Genes." Each of the above
documents is incorporated herein by reference in their
entireties.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0003] The entire content of the following electronic submission of
the sequence listing via the USPTO EFS-WEB server, as authorized
and set forth in MPEP .sctn. 1730 II.B.2(a)(C), is incorporated
herein by reference in its entirety for all purposes. The sequence
listing is identified on the electronically filed text file as
follows:
TABLE-US-00001 File Name Date of Creation Size (bytes)
300622007511Seqlist.txt Jun. 17, 2008 17,817 bytes
BACKGROUND
[0004] The clinical utility of many potential anti-cancer compounds
is limited by undesired toxicity against non-target cells.
Undesired toxicity in a drug is typically due to a lack of
specificity, either in target tissue or in mechanism of action. If
the drug target is present in normal as well as diseased tissues,
then normal tissue as well as diseased tissue may be affected by
the drug. The drug may also have multiple mechanisms of toxicity,
one specific for diseased cells and the other non-specific. In
either instance, there is often a dose-dependent difference in the
actions of drugs against normal and diseased tissues, with the
effects on diseased tissues being observed at lower concentrations
than the effects on normal tissues. A major task of anti-cancer
therapy is thus to determine the dosage at which the drug is
therapeutically effective with minimal effects on normal
tissues.
[0005] Phase I clinical trials are typically used to determine the
maximum tolerated dose (MTD) of a potential anti-cancer compound,
i.e., the maximum dose that can be safely administered without
incurring toxicity. The difference between the MTD and the
therapeutically effective dose is known as the therapeutic window.
For a large number of anti-cancer agents, the MTD is very close to
the therapeutically effective dose, i.e., the therapeutic window is
very small. The MTD may even be lower than the therapeutically
effective dose, making the agent unusable in the clinic.
[0006] Clinical anti-cancer therapy often involves attempting to
achieve a delicate balance between effectiveness and undesired
toxicity. Agents which synergize the action of a drug against
diseased tissues while not affecting the toxicity against normal
tissues could allow the effective use of doses of drug well below
the MTD, thus increasing the therapeutic window and enhancing the
safety and effectiveness of the therapy.
[0007] Geldanamycin (FIG. 1) is a benzoquinone ansamycin polyketide
isolated from Streptomyces geldanus. Although originally discovered
by screening microbial extracts for antibacterial and antiviral
activity, geldanamycin was later found to be cytotoxic to certain
tumor cells in vitro and to reverse the morphology of cells
transformed by the Rous sarcoma virus to a normal state.
[0008] Geldanamycin's nanomolar potency and apparent specificity
for aberrant protein kinase dependent tumor cells, as well as the
discovery that its primary target in mammalian cells is the
ubiquitous Hsp90 protein chaperone, has stimulated interest in the
development of this anti-cancer drug. However, the association of
hepatotoxicity with the administration of geldanamycin led to its
withdrawal from Phase I clinical trials. As with several other
promising anticancer agents, geldanamycin also has poor water
solubility that makes it difficult to deliver in therapeutically
effective doses.
[0009] More recently, attention has focused on 17-amino derivatives
of geldanamycin, in particular
17-(allylamino)-17-desmethoxygeldanamycin (17-AAG; FIG. 1), that
show reduced hepatotoxicity while maintaining Hsp90 binding.
Certain 17-amino derivatives of geldanamycin, 11-oxogeldanamycin,
and 5,6-dihydrogeldanamycin, are disclosed in U.S. Pat. Nos.
4,261,989; 5,387,584; and 5,932,566, each of which is incorporated
herein by reference. Like geldanamycin, 17-AAG has limited aqueous
solubility. This property requires the use of a solubilizing
carrier, most commonly Cremophore.RTM. (BASF Aktiengesellschaft), a
polyethoxylated castor oil which can result in serious side
reactions in some patients.
[0010] Treatment of cancer cells with geldanamycin or 17-AAG causes
a retinoblastoma protein-dependent G1 block, mediated by
down-regulation of the induction pathways for cyclin D-cyclin
dependent cdk4 and cdk6 protein kinase activity. Cell cycle arrest
is followed by differentiation and apoptosis. G1 progression is
unaffected by geldanamycin or 17-AAG in cells with mutated
retinoblastoma protein; these cells undergo cell cycle arrest after
mitosis, again followed by apoptosis.
[0011] The mechanism of action of benzoquinone ansamycins appears
to be via binding to Hsp90 and subsequent degradation of
Hsp90-associated client proteins. Among the most sensitive client
protein targets of the benzoquinone ansamycins are the Her kinases
(also known as ErbB), Raf, Met tyrosine kinase, and the steroid
receptors. Hsp90 is also involved in the cellular response to
stress, including heat, radiation, and toxins. Certain benzoquinone
ansamycins, such as 17-AAG, have thus been studied to determine
their interaction with cytotoxins that do not target Hsp90 client
proteins.
[0012] U.S. Pat. Nos. 6,245,759; 6,306,874; and 6,313,138, each of
which is incorporated herein by reference, disclose compositions
comprising certain tyrosine kinase inhibitors together with 17-AAG
and methods for treating cancer with such compositions. Munster et
al., "Modulation of Hsp90 function by ansamycins sensitizes breast
cancer cells to chemotherapy-induced apoptosis in an RB- and
schedule-dependent manner," Clinical Cancer Research (2001) 7:
2228-2236, discloses that 17-AAG sensitizes cells in culture to the
cytotoxic effects of paclitaxel and doxorubicin. The Munster
reference further discloses that the sensitization towards
paclitaxel by 17-AAG is schedule-dependent in retinoblastoma
protein-producing cells due to the action of these two drugs at
different stages of the cell cycle: treatment of cells with a
combination of paclitaxel and 17-AAG is reported to give
synergistic apoptosis, while pretreatment of cells with 17-AAG
followed by treatment with paclitaxel is reported to result in
abrogation of apoptosis. Treatment of cells with paclitaxel
followed by treatment with 17-AAG 4 hours later is reported to show
a synergistic effect similar to coincident treatment.
[0013] Citri et al., "Drug-induced ubiquitylation and degradation
of ErbB receptor tyrosine kinases: implications for cancer
chemotherapy," EMBO Journal (2002) 21: 2407-2417, discloses an
additive effect upon co-administration of geldanamycin and an
irreversible protein kinase inhibitor, CI-1033, on growth of
ErbB2-expressing cancer cells in vitro. In contrast, an
antagonistic effect of CI-1033 and an anti-ErbB2 antibody,
Herceptin, is disclosed.
[0014] Thus, while there has been a great deal of research interest
in the benzoquinone ansamycins, particularly geldanamycin and
17-AAG, there remains a need for effective therapeutic regimens to
treat cancer or other diseases or conditions characterized by
undesired cellular hyperproliferation using such compounds, whether
alone or in combination with other agents. If water-soluble
benzoquinone ansamycins were available, such compounds might be
more readily formulated and more effective in clinical treatment
without dangerous hepatotoxicity. If effective therapeutic
treatment regimens were available for administering the
benzoquinone ansamycins with other proven anti-cancer compounds,
there could be new and more effective means of treating cancer. If
the potential of using a benzoquinone ansamycin to lower the
effective dose of another anti-cancer agent could be realized, then
not only could less expensive therapies be made available (since
less drug would need to be administered) but also, and more
importantly, one could use drugs that have previously not been
useful in chemotherapy due to their narrow therapeutic window.
Thus, there is an unmet need for synergists of anti-cancer
compounds that allow for administration of doses significantly
below the maximum tolerated dose while maintaining therapeutic
effectiveness, along with appropriate dosing schedules for
combination therapy. The present invention meets such needs in that
it provides novel benzoquinone ansamycins and provides methods for
using these novel compounds as well as known compounds in
single-agent and combination therapies for the treatment of cancer
and other diseases or conditions characterized by undesired
cellular hyperproliferation.
SUMMARY OF THE INVENTION
[0015] The present invention provides compounds, methods for their
preparation and intermediates thereto, and methods for the use of
these compounds in the treatment of diseases or conditions
characterized by undesired cellular proliferation or
hyperproliferation.
[0016] In one aspect, the invention provides novel benzoquinone
ansamycins related to geldanamycin. These analogs are prepared
through chemical manipulation and/or genetic engineering. Compounds
having improved solubility properties and compounds having
conformations optimized to bind Hsp90 are also provided.
[0017] In a second aspect, the invention provides genetically
engineered forms of the geldanamycin polyketide synthase
biosynthetic gene cluster, vectors comprising said gene clusters,
host cells comprising said vectors, and methods for the production
of geldanamycin analogs using said host cells.
[0018] In a third aspect, the invention provides compositions
comprising benzoquinone ansamycins for the treatment of diseases or
conditions characterized by undesired cellular proliferation or
hyperproliferation. In certain embodiments, the disease is
cancer.
[0019] In a fourth aspect, the invention provides combination
therapies comprising the use of a benzoquinone ansamycin and a
second agent for use in the treatment of diseases or conditions
characterized by undesired cellular hyperproliferation. In certain
embodiments, the disease is cancer. In certain embodiments, the
second agent is an inhibitor of an Hsp90 client protein. In certain
embodiments, the second agent is a protein kinase inhibitor. In
certain embodiments, the second agent is a microtubule stabilizing
agent. In certain embodiments, the second agent is a cytotoxic
drug. In one embodiment, the second agent has been approved by the
Federal Drug Administration as a stand-alone agent for the
treatment of cancer. In another embodiment, the second agent has
not entered or has entered but not progressed through clinical
trials in the United States due to overt toxicity or narrow
therapeutic window.
[0020] In a fifth aspect, the invention provides methods for
preventing undesired cell adhesion and growth on devices for in
vivo use. These devices include stents, catheters, prostheses and
the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows the structures of various naturally-occurring
benzoquinone ansamycins as well as 17-AAG anf 17-DMAG.
[0022] FIG. 2 shows particular embodiments of the compounds having
formula (I) having groups with solubilizing functionalities.
[0023] FIG. 3 shows the results of treating SKBr3 cells with a
benzoquinone ansamycin and the protein kinase inhibitor Iressa
according to the methods of the present invention. Panel A shows
results with 17-AAG. Panel B shows results with 17-DMAG.
[0024] FIG. 4 shows the structures of representative protein kinase
inhibitors.
[0025] FIG. 5 shows the results of treating H358 cells with 17-AAG
and the microtubule stabilizing agent paclitaxel according to the
methods of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention provides compounds, intermediates
thereto, and methods for the use of these compounds in the
treatment of diseases or conditions characterized by undesired
cellular hyperproliferation.
[0027] Statements regarding the scope of the present invention and
definitions of terms used herein are listed below. The definitions
apply to the terms as they are used throughout this specification,
unless otherwise limited in specific instances, either individually
or as part of a larger group.
[0028] Where stereochemistry is not specifically indicated, all
stereoisomers of the inventive compounds are included within the
scope of the invention, as pure compounds as well as mixtures
thereof. Unless otherwise indicated, individual enantiomers,
diastereomers, geometrical isomers, and combinations and mixtures
thereof are all encompassed by the present invention. Polymorphic
crystalline forms and solvates are also encompassed within the
scope of this invention.
[0029] Protected forms of the inventive compounds are included
within the scope of this invention. A variety of protecting groups
are disclosed, for example, in T. H. Greene and P. G. M. Wuts,
Protective Groups in Organic Synthesis, Third Edition, John Wiley
& Sons, New York (1999). The present invention includes within
its scope prodrugs of the active compounds of this invention. Such
prodrugs are in general functional derivatives of the compounds
that are readily convertible in vivo into the required compound.
Thus, in the methods of treatment of the present invention, the
term "administering" shall encompass the treatment of the various
disorders described with the compound specifically disclosed or
with a compound which may not be specifically disclosed, but which
converts to the specified compound in vivo after administration to
a subject in need thereof. Conventional procedures for the
selection and preparation of suitable prodrug derivatives are
described, for example, in "Design of Prodrugs," H. Bundgaard ed.,
Elsevier, 1985.
[0030] As used herein, the terms "benzoquinone ansamycin" refers to
a compound comprising a benzoquinone nucleus connected at two
non-adjacent positions by a macrocyclic lactam. Specific examples
of naturally-occurring benzoquinone ansamycins include but are not
limited to geldanamycin, herbimycin, macbecin, mycotrienes, and
ansamitocin. The term "geldanamycin analog" refers to a type of
benzoquinone ansamycin that can be derived from geldanamycin by
chemical manipulation or by manipulation of the geldanamycin
biosynthetic gene cluster, such as
17-allylamino-17-desmethoxygeldanamycin (17-AAG),
17-(2-dimethylaminoethyl)amino-17-desmethoxygeldanamycin (17-DMAG),
or a compound having a structure shown in formula (I).
[0031] As used herein, the term "aliphatic" refers to saturated and
non-aromatic unsaturated straight chain, branched chain, cyclic, or
polycyclic hydrocarbons. Illustrative examples of aliphatic groups
include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and
cycloalkynyl groups. The term "alkyl" refers to a straight or
branched chain saturated hydrocarbon substituent. "Alkenyl" refers
to a straight or branched chain hydrocarbon substituent with at
least one carbon-carbon double bond. "Alkynyl" refers to a straight
or branched chain hydrocarbon substituent with at least one
carbon-carbon triple bond.
[0032] The term "aryl" refers to monocyclic or polycyclic groups
having at least one aromatic ring structure that include preferably
one to fourteen carbon atoms. Illustrative examples of aryl groups
include but are not limited to: naphthyl, phenyl,
tetrahydronaphthyl, and the like.
[0033] The term "heteroaryl" refers to monocyclic or polycyclic
groups having at least one aromatic ring structure and that include
one or more heteroatoms and preferably one to fourteen carbon
atoms. Illustrative examples of heteroaryl groups include but are
not limited to: furanyl, imidazolyl, indanyl, indolyl, indazolyl,
isoxazolyl, isoquinolyl, oxazolyl, oxadiazolyl, pyrazinyl, pyridyl,
pyrimidinyl, pyrrolyl, pyrazolyl, quinolyl, quinoxalyl, tetrazolyl,
thiazolyl, thienyl, and the like.
[0034] The aliphatic, aryl, and heteroaryl moieties may be
substituted with one or more substituents, preferably from one to
five substituents, more preferably from one to three substituents,
and most preferably from one to two substituents, and as such are
referred to as "substituted aliphatic," "substituted aryl," and
"substituted heteroaryl." The definition of any substituent or
variable at a particular location in a molecule is independent of
its definitions elsewhere in that molecule. It is understood that
substituents and substitution patterns on the compounds of this
invention can be selected by one of ordinary skill in the art to
provide compounds that are chemically stable and that can be
readily synthesized by techniques known in the art as well as those
methods set forth herein. Examples of suitable substituents include
but are not limited to: aliphatic, haloaliphatic, halogen, aryl,
heteroaryl, hydroxy, alkoxy, aryloxy, azido, thio, alkylthio,
arylthio, amino, alkylamino, arylamino, acyl, carbamoyl,
sulfonamido, nitro, cyano, carboxy, guanidine, and the like.
[0035] The term "haloaliphatic" refers to a substituted aliphatic
group substituted by one or more halogens.
[0036] The terms "halo, "halogen," or "halide" refer to fluorine,
chlorine, bromine, and iodine.
[0037] The term "acyl" refers to --C(.dbd.O)R, where R is an
aliphatic group.
[0038] The term "alkoxy" refers to --OR, where R is an aliphatic
group.
[0039] The term "aryloxy" refers to --OR, where R is an aryl
group.
[0040] The term "carbamoyl" refers to --O(C.dbd.O)NRR', where R and
R' are independently H, aliphatic, or aryl groups.
[0041] The term "alkylamino" refers to --NHR, where R is an alkyl
group. The term "dialkylamino" refers to --NRR', where both R and
R' are alkyl groups.
[0042] The term "hydroxyalkyl" refers to --R--OH, where R is an
aliphatic group.
[0043] The term "aminoalkyl" refers to --R--NH.sub.2, where R is an
aliphatic group. The term "alkylaminoalkyl" refers to --R--NH--R',
where both R and R' are aliphatic groups. The term
"dialkylaminoalkyl" refers to --R--N(R')--R'', where R, R', and R''
are aliphatic groups. The term "arylaminoalkyl" refers to
--R--NH--R', where R is an aliphatic and R' is an aryl group.
[0044] The term "oxo" refers to a carbonyl oxygen (.dbd.O).
[0045] The term "isolated" as used herein means that the isolated
material is in a preparation in which said material forms a major
component of the preparation, such as constituting about 50%, about
60%, about 70%, about 80%, about 90%, about 95%, about 99%, or more
by weight of the components in the preparation.
[0046] The term "subject" as used herein, refers to an animal,
typically a mammal or a human, that has been the object of
treatment, observation, and/or experiment. When the term is used in
conjunction with administration of a compound or drug, then the
subject has been the object of treatment, observation, and/or
administration of the compound or drug.
[0047] The term "therapeutically effective amount" as used herein,
means that amount of active compound or pharmaceutical agent that
elicits the biological or medicinal response in a cell culture,
tissue system, animal, or human that is being sought by a
researcher, veterinarian, clinician, or physician, which includes
alleviation of the symptoms of the disease, condition, or disorder
being treated.
[0048] The term "composition" is intended to encompass a product
comprising the specified ingredients in the specified amounts, as
well as any product that results, directly or indirectly, from
combinations of the specified ingredients in the specified
amounts.
[0049] The term "pharmaceutically acceptable salt" refers to a salt
of one or more compounds. Suitable pharmaceutically acceptable
salts of compounds include acid addition salts which may, for
example, be formed by mixing a solution of the compound with a
solution of a pharmaceutically acceptable acid such as hydrochloric
acid, hydrobromic acid, sulfuric acid, fumaric acid, maleic acid,
succinic acid, benzoic acid, acetic acid, citric acid, tartaric
acid, phosphoric acid, carbonic acid, or the like. Where the
compounds carry one or more acidic moieties, pharmaceutically
acceptable salts may be formed by treatment of a solution of the
compound with a solution of a pharmaceutically acceptable base,
such as lithium hydroxide, sodium hydroxide, potassium hydroxide,
tetraalkylammonium hydroxide, lithium carbonate, sodium carbonate,
potassium carbonate, ammonia, alkylamines, or the like.
[0050] The term "pharmaceutically acceptable carrier" refers to a
medium that is used to prepare a desired dosage form of a compound.
A pharmaceutically acceptable carrier can include one or more
solvents, diluents, or other liquid vehicles; dispersion or
suspension aids; surface active agents; isotonic agents; thickening
or emulsifying agents; preservatives; solid binders; lubricants;
and the like. Remington's Pharmaceutical Sciences, Fifteenth
Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1975) and
Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe
ed. (American Pharmaceutical Assoc. 2000), disclose various
carriers used in formulating pharmaceutical compositions and known
techniques for the preparation thereof.
[0051] The term "pharmaceutically acceptable ester" refers to an
ester that hydrolyzes under physiologically relevant conditions to
produce a compound or a salt thereof. Illustrative examples of
suitable ester groups include but are not limited to formates,
acetates, propionates, butyrates, succinates, and
ethylsuccinates.
[0052] The term "client protein" refers to a protein that interacts
with a chaperone, for example Hsp90. In one aspect, this
interaction with a chaperone is useful or required either for
proper folding or for stabilization and maintenance. In another
aspect, the chaperone forms the core of a functional receptor
complex. In both of these aspects, the interaction with the
chaperone may be direct or mediated through one or more other
proteins. Table 1 below provides an illustrative list of the client
proteins of Hsp90. The term "clientele" refers to the complete set
of client proteins for a chaperone.
TABLE-US-00002 TABLE 1 Illustrative list of Hsp90 client proteins
Client protein Function Steroid Hormone Receptors Glucocorticoid
receptor ligand-mediated gene transcription Estrogen receptor
ligand-mediated gene transcription Androgen receptor
ligand-mediated gene transcription Progesterone receptor
ligand-mediated gene transcription Protein Kinases c-SRC, v-SRC
signal transduction LCK T-cell development &function WEE1 Cell
cycle regulation (G2) MYT1 Cell cycle regulation (G2) ErbB2 (Her-2)
Signal transduction EGFR (ErbB1) Signal transduction FPS/FES Cell
proliferation c-RAF-1, v-RAF-1 MAPK signaling MEK MAPK signaling
Casein kinase 2 pleiotropic kinase CDK4 Cell cycle regulation (G1)
AKT (PKB) PI3 kinase signaling Death domain kinase RIP TNF-mediated
necrosis Bcr-ABL myeloid leukemia pathogenesis PIM-1
cytokine-mediated proliferation MOK MAPK signaling Polo-1 kinase
(PLK) Cell cycle regulation (G2/M) Focal adhesion kinase (FAK)
actin-based cell motility c-MET HGF/SF-MET motility signaling eIF2
kinase transcriptional regulation Other Client Proteins Mutant p53
cell cycle checkpoint protein mutant Hepatitis B reverse
transcriptase viral transcription hTERT (telomerase subunit) cell
mortality, senescence .beta..gamma. subunits of trimeric G proteins
signal transduction endothelial NOS NO synthesis calcineurin
Ca.sup.2+-dependent signaling tubulin microtubule formation
HIF-1.alpha. hypoxia-induced angiogenesis Retinoblastoma protein
cell cycle regulation (G1/S) Tumor necrosis factor receptor 1
TNF-mediated apoptosis Cystic fibrosis transmembrane Ion
channel/cystic fibrosis conductance regulator Immunoglobulin chains
Immune response Fanconi anemia protein hematopoiesis Apoprotein B
atherosclerosis Aryl hydrocarbon receptor gene transcription SV40 T
antigen viral oncogene
[0053] In one aspect of the invention, geldanamycin analogs having
the formula (I) are provided:
##STR00001##
wherein R.sup.1 is MeO, (CH.sub.2).sub.3N or R.sup.9--NH, wherein
R.sup.9 is selected from the group consisting of H, substituted or
unsubstituted C.sub.1-C.sub.6 alkyl, substituted or unsubstituted
C.sub.1-C.sub.6 alkenyl, substituted or unsubstituted
C.sub.1-C.sub.6 alkynyl, substituted or unsubstituted
C.sub.3-C.sub.6 cycloalkyl, piperidinyl, N-alkylpiperidinyl,
hexahydropyranyl, furfuryl, tetrahydrofurfuryl, pyrrolidinyl,
N-alkylpyrrolidinyl, piperazinylamino, N-alkylpiperazinyl,
morpholinyl, N-alkylaziridinylmethyl,
(1-azabicyclo[1.3.0]hex-1-yl)ethyl,
2-(N-methyl-pyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl,
2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl,
2-(4-pyridyl)ethyl, and 3-(4-morpholino)-1-propyl, or R.sup.6 is H
and R.sup.1 and R.sup.5 taken together form a group of the formula
NH-Z-O, wherein Z is a linker comprised of from 1 to 6 carbon atoms
and 0 to 2 nitrogen atoms and wherein the 0 is attached at the
position of R.sup.5; R.sup.2 is selected from the group consisting
of H, halogen, OR.sup.10, NHR.sup.10, SR.sup.10, aryl, and
heteroaryl, wherein R.sup.10 is selected from the group consisting
of substituted or unsubstituted C.sub.1-C.sub.6 alkyl, substituted
or unsubstituted C.sub.1-C.sub.6 alkenyl, substituted or
unsubstituted C.sub.1-C.sub.6 alkynyl, and substituted or
unsubstituted C.sub.3-C.sub.6 cycloalkyl; R.sup.3 is H, OH, or OMe;
R.sup.4 is H or Me; R.sup.5 is OH or O--C(.dbd.O)--CH.sub.2NH.sub.2
and R.sup.6 is H, or R.sup.5 and R.sup.6 taken together form .dbd.O
or .dbd.N--OR.sup.11, wherein R.sup.11 is selected from the group
consisting of H, substituted or unsubstituted C.sub.1-C.sub.6
alkyl, substituted or unsubstituted C.sub.1-C.sub.6 alkenyl,
substituted or unsubstituted C.sub.1-C.sub.6 alkynyl, substituted
or unsubstituted C.sub.3-C.sub.6 cycloalkyl, aryl, or heteroaryl;
R.sup.7 is H and R.sup.8 is H or OH, or R.sup.7 and R.sup.8 taken
together form a bond; and X is O or a bond, with the provisos that
when R.sup.3 is H, R.sup.4 is Me, and R.sup.7 is H and R.sup.8 is H
or R.sup.7 and R.sup.8 taken together form a bond that either
R.sup.6 is H and R.sup.1 and R.sup.5 taken together form a group of
the formula NH-Z-O, wherein Z is a linker comprised of from 1 to 6
carbon atoms and 0 to 2 nitrogen atoms and wherein the 0 is
attached at the position of R.sup.5, or that R.sup.1 is
(CH.sub.2).sub.3N or R.sup.9--NH, wherein R.sup.9 is selected from
the group consisting of piperidinyl, N-alkylpiperidinyl,
hexahydropyranyl, furfuryl, tetrahydrofurfuryl, pyrrolidinyl,
N-alkylpyrrolidinyl, piperazinylamino, N-alkylpiperazinyl,
morpholinyl, N-alkylaziridinylmethyl,
(1-azabicyclo[1.3.0]hex-1-yl)ethyl,
2-(N-methyl-pyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl,
2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl,
2-(4-pyridyl)ethyl, and 3-(4-morpholino)-1-propyl; and that when
R.sup.3 is H and R.sup.4 is Me that R.sup.7 is H and R.sup.8 is
OH.
[0054] In one embodiment, compounds having formula (I) are provided
wherein R.sup.1 is (CH.sub.2).sub.3N or R.sup.9--NH, wherein
R.sup.9 is selected from the group consisting of piperidinyl,
N-alkylpiperidinyl, hexahydropyranyl, furfuryl, tetrahydrofurfuryl,
pyrrolidinyl, N-alkylpyrrolidinyl, piperazinylamino,
N-alkylpiperazinyl, morpholinyl, N-alkylaziridinylmethyl,
(1-azabicyclo[1.3.0]hex-1-yl)ethyl,
2-(N-methyl-pyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl,
2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl,
2-(4-pyridyl)ethyl, and 3-(4-morpholino)-1-propyl; R.sup.2 is
selected from the group consisting of H, halogen, OR.sup.10,
NHR.sup.10, SR.sup.10, aryl, and heteroaryl, wherein R.sup.10 is
selected from the group consisting of substituted or unsubstituted
C.sub.1-C.sub.6 alkyl, substituted or unsubstituted C.sub.1-C.sub.6
alkenyl, substituted or unsubstituted C.sub.1-C.sub.6 alkynyl, and
substituted or unsubstituted C.sub.3-C.sub.6 cycloalkyl; R.sup.3 is
H, OH, or OMe; R.sup.4 is H or Me; R.sup.5 is OH or
O--C(.dbd.O)--CH.sub.2NH.sub.2 and R.sup.6 is H, or R.sup.5 and
R.sup.6 taken together form .dbd.O or .dbd.N--OR.sup.11, wherein
R.sup.11 is selected from the group consisting of H, substituted or
unsubstituted C.sub.1-C.sub.6 alkyl, substituted or unsubstituted
C.sub.1-C.sub.6 alkenyl, substituted or unsubstituted
C.sub.1-C.sub.6 alkynyl, substituted or unsubstituted
C.sub.3-C.sub.6 cycloalkyl, aryl, or heteroaryl; R.sup.7 is H and
R.sup.8 is H or OH, or R.sup.7 and R.sup.8 taken together form a
bond; and X is O or a bond.
[0055] In one embodiment, compounds having formula (I) are provided
wherein R.sup.1 is (CH.sub.2).sub.3N or R.sup.9--NH, wherein
R.sup.9 is selected from the group consisting of piperidinyl,
N-alkylpiperidinyl, hexahydropyranyl, furfuryl, tetrahydrofurfuryl,
pyrrolidinyl, N-alkylpyrrolidinyl, piperazinylamino,
N-alkylpiperazinyl, morpholinyl, N-alkylaziridinylmethyl,
(1-azabicyclo[1.3.0]hex-1-yl)ethyl,
2-(N-methyl-pyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl,
2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl,
2-(4-pyridyl)ethyl, and 3-(4-morpholino)-1-propyl; R.sup.2 is H;
R.sup.3 is H, OH, or OMe; R.sup.4 is H or Me; R.sup.5 is OH or
O--C(.dbd.O)--CH.sub.2NH.sub.2 and R.sup.6 is H, or R.sup.5 and
R.sup.6 taken together form .dbd.O or .dbd.N--OR.sup.11, wherein
R.sup.11 is selected from the group consisting of H, substituted or
unsubstituted C.sub.1-C.sub.6 alkyl, substituted or unsubstituted
C.sub.1-C.sub.6 alkenyl, substituted or unsubstituted
C.sub.1-C.sub.6 alkynyl, substituted or unsubstituted
C.sub.3-C.sub.6 cycloalkyl, aryl, or heteroaryl; R.sup.7 is H and
R.sup.8 is H or OH, or R.sup.7 and R.sup.8 taken together form a
bond; and X is O or a bond.
[0056] In one embodiment, geldanamycin analogs having improved
solubility are provided resulting from chemical manipulation of
geldanamycin to provide compounds having formula (I) wherein:
R.sup.1 is (CH.sub.2).sub.3N or R.sup.9--NH, wherein R.sup.9 is
selected from the group consisting of H, substituted or
unsubstituted C.sub.1-C.sub.6 alkyl, substituted or unsubstituted
C.sub.1-C.sub.6 alkenyl, substituted or unsubstituted
C.sub.1-C.sub.6 alkynyl, substituted or unsubstituted
C.sub.3-C.sub.6 cycloalkyl, piperidinyl, N-alkylpiperidinyl,
hexahydropyranyl, furfuryl, tetrahydrofurfuryl, pyrrolidinyl,
N-alkylpyrrolidinyl, piperazinylamino, N-alkylpiperazinyl,
morpholinyl, N-alkylaziridinylmethyl,
(1-azabicyclo[1.3.0]hex-1-yl)ethyl,
2-(N-methyl-pyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl,
2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl,
2-(4-pyridyl)ethyl, and 3-(4-morpholino)-1-propyl; R.sup.2 is
selected from the group consisting of H, halogen, OR.sup.10,
NHR.sup.10, SR.sup.10, aryl, and heteroaryl, wherein R.sup.10 is
selected from the group consisting of substituted or unsubstituted
C.sub.1-C.sub.6 alkyl, substituted or unsubstituted C.sub.1-C.sub.6
alkenyl, substituted or unsubstituted C.sub.1-C.sub.6 alkynyl, and
substituted or unsubstituted C.sub.3-C.sub.6 cycloalkyl; R.sup.3 is
H; R.sup.4 is methyl; R.sup.5 is OH or
O--C(.dbd.O)--CH.sub.2NH.sub.2 and R.sup.6 is H, or R.sup.5 and
R.sup.6 taken together form .dbd.O or .dbd.N--OH; R.sup.7 and
R.sup.8 are H, or R.sup.7 and R.sup.8 taken together form a bond;
and X is O or a bond.
[0057] In another embodiment of the invention, compounds having
formula (I) are provided wherein: R.sup.1 is R.sup.9--NH, wherein
R.sup.9 is selected from the group consisting of ethyl,
2-(dimethylamino)ethyl, 2-fluoroethyl, 2,2-difluoroethyl,
2-(N-methylpyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl,
2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl,
2-(4-pyridyl)ethyl, 3-(4-morpholino)-1-propyl,
3-(dimethylamino)-1-propyl, 3-(dimethylamino)-2-propyl,
2-(dimethylamino)-1-propyl, and cyclopropyl-methyl; R.sup.2 is H;
R.sup.3 is H; R.sup.4 is methyl; R.sup.5 is OH or
O--C(.dbd.O)--CH.sub.2NH.sub.2 and R.sup.6 is H, or R.sup.5 and
R.sup.6 taken together form .dbd.O or .dbd.N--OH; R.sup.7 and
R.sup.8 are H, or R.sup.7 and R.sup.8 taken together form a bond;
and X is O or a bond.
[0058] In another embodiment of the invention, compounds having the
structures:
##STR00002##
are provided.
[0059] In another embodiment, geldanamycin analogs having formula
(I) are provided wherein R.sup.1 is OMe; R.sup.2 is H; R.sup.3 is
H, OH, or OMe; R.sup.4 is H or methyl; R.sup.5 is OH and R.sup.6 is
H; R.sup.7 is H and R.sup.8 is H or OH, or R.sup.7 and R.sup.8
taken together form a bond; and X is a bond, with the proviso that
geldanamycin and 4,5-dihydrogeldanamycin are not included.
[0060] Other embodiments of the invention provide compounds having
the formulas:
##STR00003##
[0061] In other embodiments of the invention, the geldanamycin
analogs described above serve as starting materials for chemical
addition of solubilizing groups. In one embodiment,
15-hydroxygeldanamycin is derivatized to provide compounds having
formula (I) wherein: R.sup.1 is (CH.sub.2).sub.3N or R.sup.9--NH,
wherein R.sup.9 is selected from the group consisting of H,
substituted or unsubstituted C.sub.1-C.sub.6 alkyl, substituted or
unsubstituted C.sub.1-C.sub.6 alkenyl, substituted or unsubstituted
C.sub.1-C.sub.6 alkynyl, substituted or unsubstituted
C.sub.3-C.sub.6 cycloalkyl, piperidinyl, N-alkylpiperidinyl,
hexahydropyranyl, furfuryl, tetrahydro-furfuryl, pyrrolidinyl,
N-alkylpyrrolidinyl, piperazinylamino, N-alkylpiperazinyl,
morpholinyl, N-alkylaziridinylmethyl,
(1-azabicyclo[1.3.0]hex-1-yl)ethyl,
2-(N-methyl-pyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl,
2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl,
2-(4-pyridyl)ethyl, and 3-(4-morpholino)-1-propyl; R.sup.3 is OH;
R.sup.4 is methyl; R.sup.5 is OH or O--C(.dbd.O)--CH.sub.2NH.sub.2
and R.sup.6 is H, or R.sup.5 and R.sup.6 taken together form .dbd.O
or .dbd.N--OH; and R.sup.7 and R.sup.8 taken together form a bond;
and X is O or a bond.
[0062] In another embodiment of the invention,
15-hydroxygeldanamycin is derivatized to provide compounds having
formula (I) wherein: R.sup.1 is (CH.sub.2).sub.3N or R.sup.9--NH,
wherein R.sup.9 is selected from the group consisting of allyl,
ethyl, 2-(dimethylamino)ethyl, 2-fluoroethyl, 2,2-difluoroethyl,
2-(N-methylpyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl,
2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl,
2-(4-pyridyl)ethyl, 3-(4-morpholino)-1-propyl,
3-(dimethylamino)-1-propyl, 3-(dimethylamino)-2-propyl,
2-(dimethylamino)-1-propyl, and cyclopropylmethyl; R.sup.2 is H;
R.sup.3 is OH; R.sup.4 is methyl; R.sup.5 is OH or
O--C(.dbd.O)--CH.sub.2NH.sub.2 and R.sup.6 is H, or R.sup.5 and
R.sup.6 taken together form .dbd.O or .dbd.N--OH; R.sup.7 and
R.sup.8 taken together form a bond; and X is O or a bond.
[0063] In another embodiment, 15-hydroxygeldanamycin analogs having
the formulas
##STR00004##
are provided.
[0064] In one embodiment, 28-desmethylgeldanamycin is derivatized
to provide compounds having formula (I) wherein: R.sup.1 is
(CH.sub.2).sub.3N or R.sup.9--NH, wherein R.sup.9 is selected from
the group consisting of H, substituted or unsubstituted
C.sub.1-C.sub.6 alkyl, substituted or unsubstituted C.sub.1-C.sub.6
alkenyl, substituted or unsubstituted C.sub.1-C.sub.6 alkynyl,
substituted or unsubstituted C.sub.3-C.sub.6 cycloalkyl,
piperidinyl, N-alkylpiperidinyl, hexahydropyranyl, furfuryl,
tetrahydro-furfuryl, pyrrolidinyl, N-alkylpyrrolidinyl,
piperazinylamino, N-alkylpiperazinyl, morpholinyl,
N-alkylaziridinylmethyl, (1-azabicyclo[1.3.0]hex-1-yl)ethyl,
2-(N-methylpyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl,
2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl,
2-(4-pyridyl)ethyl, and 3-(4-morpholino)-1-propyl; R.sup.2 is
selected from the group consisting of H, halogen, OR.sup.10,
NHR.sup.10, SR.sup.10, aryl, and heteroaryl, wherein R.sup.10 is
selected from the group consisting of substituted or unsubstituted
C.sub.1-C.sub.6 alkyl, substituted or unsubstituted C.sub.1-C.sub.6
alkenyl, substituted or unsubstituted C.sub.1-C.sub.6 alkynyl, and
substituted or unsubstituted C.sub.3-C.sub.6 cycloalkyl; R.sup.3 is
H; R.sup.4 is H; R.sup.5 is OH or O--C(.dbd.O)--CH.sub.2NH.sub.2
and R.sup.6 is H, or R.sup.5 and R.sup.6 taken together form .dbd.O
or .dbd.N--OH; and R.sup.7 and R.sup.8 taken together form a bond;
and X is O or a bond.
[0065] In another embodiment of the invention,
28-desmethylgeldanamycin is derivatized to provide compounds having
formula (I) wherein: R.sup.1 is (CH.sub.2).sub.3N or R.sup.9--NH,
wherein R.sup.9 is selected from the group consisting of allyl,
ethyl, 2-(dimethylamino)ethyl, 2-fluoroethyl, 2,2-difluoroethyl,
2-(N-methylpyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl,
2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl,
2-(4-pyridyl)ethyl, 3-(4-morpholino)-1-propyl,
3-(dimethylamino)-1-propyl, 3-(dimethylamino)-2-propyl,
2-(dimethylamino)-1-propyl, and cyclopropylmethyl; R.sup.2 is H;
R.sup.3 is H; R.sup.4 is H; R.sup.5 is OH or
O--C(.dbd.O)--CH.sub.2NH.sub.2 and R.sup.6 is H, or R.sup.5 and
R.sup.6 taken together form .dbd.O or .dbd.N--OH; R.sup.7 and
R.sup.8 taken together form a bond; and X is O or a bond.
[0066] In another embodiment, 28-desmethylgeldanamycin analogs
having the formulas
##STR00005##
are provided.
[0067] In another embodiment, 4,5-dihydro-5-hydroxygeldanamycin is
derivatized to provide compounds having formula (I) wherein:
R.sup.1 is (CH.sub.2).sub.3N or R.sup.9--NH, wherein R.sup.9 is
selected from the group consisting of H, substituted or
unsubstituted C.sub.1-C.sub.6 alkyl, substituted or unsubstituted
C.sub.1-C.sub.6 alkenyl, substituted or unsubstituted
C.sub.1-C.sub.6 alkynyl, substituted or unsubstituted
C.sub.3-C.sub.6 cycloalkyl, piperidinyl, N-alkylpiperidinyl,
hexahydropyranyl, furfuryl, tetrahydro-furfuryl, pyrrolidinyl,
N-alkylpyrrolidinyl, piperazinylamino, N-alkylpiperazinyl,
morpholinyl, N-alkylaziridinylmethyl,
(1-azabicyclo[1.3.0]hex-1-yl)ethyl,
2-(N-methylpyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl,
2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl,
2-(4-pyridyl)ethyl, and 3-(4-morpholino)-1-propyl; R.sup.2 is
selected from the group consisting of H, halogen, OR.sup.10,
NHR.sup.10, SR.sup.10, aryl, and heteroaryl, wherein R.sup.10 is
selected from the group consisting of substituted or unsubstituted
C.sub.1-C.sub.6 alkyl, substituted or unsubstituted C.sub.1-C.sub.6
alkenyl, substituted or unsubstituted C.sub.1-C.sub.6 alkynyl, and
substituted or unsubstituted C.sub.3-C.sub.6 cycloalkyl; R.sup.3 is
H; R.sup.4 is methyl; R.sup.5 is OH or
O--C(.dbd.O)--CH.sub.2NH.sub.2 and R.sup.6 is H, or R.sup.5 and
R.sup.6 taken together form .dbd.O or .dbd.N--OH; and R.sup.7 is H;
R.sup.8 is OH; and X is O or a bond.
[0068] In another embodiment of the invention,
4,5-dihydro-5-hydroxygeldanamycin is derivatized to provide
compounds having formula (I) wherein: R.sup.1 is (CH.sub.2).sub.3N
or R.sup.9--NH, wherein R.sup.9 is selected from the group
consisting of allyl, ethyl, 2-(dimethylamino)ethyl, 2-fluoroethyl,
2,2-difluoroethyl, 2-(N-methylpyrrolidin-2-yl)ethyl,
2-(4-imidazolyl)ethyl, 2-(1-methyl-4-imidazolyl)ethyl,
2-(1-methyl-5-imidazolyl)ethyl, 2-(4-pyridyl)ethyl,
3-(4-morpholino)-1-propyl, 3-(dimethylamino)-1-propyl,
3-(dimethylamino)-2-propyl, 2-(dimethylamino)-1-propyl, and
cyclopropylmethyl; R.sup.2 is H; R.sup.3 is H; R.sup.4 is methyl;
R.sup.5 is OH or O--C(.dbd.O)--CH.sub.2NH.sub.2 and R.sup.6 is H,
or R.sup.5 and R.sup.6 taken together form .dbd.O or .dbd.N--OH;
R.sup.7 is H; R.sup.8 is OH; and X is O or a bond.
[0069] In another embodiment, 4,5-dihydro-5-hydroxygeldanamycin
analogs having the formulas
##STR00006##
are provided.
[0070] In another aspect of the invention, conformationally
constrained geldanamycin analogs are provided. In one embodiment,
compounds having the formula (I) are provided wherein R.sup.1 and
R.sup.5 taken together form a group of the formula NH-Z-O, wherein
Z is a linker comprised of from 1 to 6 carbon atoms and 0 to 2
nitrogen atoms and wherein the 0 is attached at the position of
R.sup.5; R.sup.2 is selected from the group consisting of H,
halogen, OR.sup.10, NHR.sup.10, SR.sup.10, aryl, and heteroaryl,
wherein R.sup.10 is selected from the group consisting of
substituted or unsubstituted C.sub.1-C.sub.6 alkyl, substituted or
unsubstituted C.sub.1-C.sub.6 alkenyl, substituted or unsubstituted
C.sub.1-C.sub.6 alkynyl, and substituted or unsubstituted
C.sub.3-C.sub.6 cycloalkyl; R.sup.3 is H or OH; R.sup.4 is H or
methyl; R.sup.7 is H and R.sup.8 is H or OH, or R.sup.7 and R.sup.8
taken together form a bond; and X is O or a bond.
[0071] In one embodiment of the invention, compounds having formula
(I) are provided wherein R.sup.1 and R.sup.5 taken together form a
group of the formula NH--(CH.sub.2).sub.4--O,
NH--CH.sub.2CH.dbd.CHCH.sub.2--O, or NH--CH.sub.2CCCH.sub.2--O;
R.sup.2 is H; R.sup.3 is H, OH, or OMe; R.sup.4 is H or methyl;
R.sup.7 is H and R.sup.8 is H or OH, or R.sup.7 and R.sup.8 taken
together form a bond; and X is O or a bond.
[0072] In another embodiment of the invention, compounds having
formula (I) are provided wherein R.sup.1 and R.sup.5 taken together
form a group of the formula NH--CH.sub.2CH.dbd.CHCH.sub.2--O;
R.sup.2 is H; R.sup.3 is H, OH, or OMe; R.sup.4 is H or methyl;
R.sup.7 is H and R.sup.8 is H or OH, or R.sup.7 and R.sup.8 taken
together form a bond; and X is O or a bond.
[0073] In another embodiment of the invention, compounds having
formula (I) are provided having the structures:
##STR00007##
[0074] In another embodiment, the water-soluble analogs described
above are subjected to conformational constraint to provide
geldanamycin analogs having both improved solubility and improved
specificity for Hsp90. Poor water solubility is a major factor
limiting the clinical usefulness of geldanamycin and 17-AAG.
Improvements in water solubility of a compound can be achieved
according to the methods of the present invention either by
addition of groups containing solubilizing functionalities to the
compound or by removal of hydrophobic groups from the compound, so
as to decrease the lipophilicity of the compound. Typical groups
containing solubilizing functionalities are shown in FIG. 2 and
include but are not limited to: 2-(dimethylaminoethyl)amino,
piperidinyl, N-alkylpiperidinyl, hexahydropyranyl, furfuryl,
tetrahydrofurfuryl, pyrrolidinyl, N-alkylpyrrolidinyl,
piperazinylamino, N-alkylpiperazinyl, morpholinyl,
N-alkylaziridinylmethyl, (1-azabicyclo[1.3.0]hex-1-yl)ethyl,
2-(N-methylpyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl,
2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl,
2-(4-pyridyl)ethyl, and 3-(4-morpholino)-1-propyl.
[0075] Solubilizing groups are added to the geldanamycin analog by
reaction of geldanamycin with amines, which results in the
displacement of the 17-methoxy group by the amine as illustrated in
Scheme 1 and exemplified in Example 1 (Schnur et al. (1995)
"Inhibition of the oncogene product p185.sup.erbB-2 in Vitro and in
Vivo by Geldanamycin and Dihydrogeldanamycin Derivatives,", J. Med.
Chem. 38, 3806-3812; Schnur et al. (1995) "erbB-2 Oncogene
Inhibition by Geldanamycin Derivatives: Synthesis, mechanism of
Action, and Structure-Activity relationships," J. Med. Chem. 38,
3813-3820; Schnur et al., "Ansamycin derivatives as antioncogene
and anticancer agents," U.S. Pat. No. 5,932,655; all of which are
incorporated herein by reference). Typical amines containing
solubilizing functionalities include 2-(dimethylamino)-ethylamine,
4-aminopiperidine, 4-amino-1-methylpiperidine,
4-aminohexahydropyran, furfurylamine, tetrahydrofurfurylamine,
3-(aminomethyl)-tetrahydrofuran, 2-(amino-methyl)pyrrolidine,
2-(aminomethyl)-1-methylpyrrolidine, 1-methylpiperazine,
morpholine, 1-methyl-2(aminomethyl)aziridine,
1-(2-aminoethyl)-1-azabicyclo-[1.3.0]hexane,
1-(2-aminoethyl)piperazine, 4-(2-aminoethyl)morpholine,
1-(2-amino-ethyl)pyrrolidine, 2-(2-aminoethyl)pyridine,
2-fluoroethylamine, 2,2-difluoroethylamine, and the like.
##STR00008##
[0076] Similar solubilizing groups can be introduced by treatment
of 19-bromo-geldanamycin or analogs with an amine containing a
solubilizing substituent in accordance with the methods of the
present invention, resulting in a 19-amino-substituted geldanamycin
analog. The 19-bromo derivative is formed upon treatment of the
geldanamycin analog with a suitable brominating reagent, such as
pyridinium bromide perbromide (Schnur et al. 1995, J. Med. Chem.
38, 3806-3812; incorporated herein by reference). Reaction of
19-bromogeldanamycin with an arylboronic acid in the presence of a
palladium catalyst according to the method of the present invention
gives 19-aryl substituted geldanamycins as illustrated in Scheme 2.
Boronic acids such as phenylboronic acid,
(4-dimethyl-amino)phenyl-boronic acid, 4-pyridylboronic acid,
4-methoxyphenylboronic acid, 2-furylboronic acid, and
4-(dimethylaminomethyl)-phenylboronic acid and the like can also be
used.
##STR00009##
[0077] In another embodiment of the invention, the geldanamycin
analogs are oxidized to produce the corresponding
11-oxogeldanamycin analogs as illustrated in Scheme 3.
##STR00010##
The 11-oxogeldanamycin analog resulting from oxidation of the 11-OH
are converted into the 11-oximino analogs by reaction with
hydroxylamine or an alkyoxylamine.
[0078] In another embodiment of the invention, the geldanamycin
analog is treated with a peroxyacid, for example
3-chloroperoxybenzoic acid (mCPBA), to produce the 8,9-epoxide, as
illustrated in Scheme 3.
[0079] In another aspect, the invention provides genetically
engineered forms of the geldanamycin polyketide synthase
biosynthetic gene cluster, vectors comprising said gene clusters,
host cells comprising said vectors, and methods for the production
of geldanamycin analogs using said host cells.
[0080] In one embodiment of the invention, substitution of the
acyltransferase domain in module 1 of the geldanamycin PKS gene
with one specific for malonyl-CoA instead of 2-methylmalonyl-CoA
results in formation of 28-desmethyl-geldanamycin. The domain swap
is created by introducing a malonyl-CoA specific acyltransferase
domain from a heterologous PKS gene, for example from the
rapamycin, tylosin, or FK520 PKS genes or the like, into the
geldanamycin PKS locus by homologous recombination into a strain
which produces geldanamycin, aided by a selectable antibiotic
resistance gene, then isolating the recombinants resulting from
double crossover events in which the wild-type acyltransferase
domain is replaced with one specific for malonyl-CoA. Details of
this are provided below in Example 5.
[0081] In another embodiment of the invention, the acyltransferase
domain in module 1 of the geldanamycin PKS gene is mutagenized
according to the methods described in Reeves et al., "Alteration of
the substrate specificity of a modular polyketide synthase
acyltransferase domain through site-directed mutagenesis,"
Biochemistry 2001, 40: 15464-15470, and in U.S. patent application
Ser. No. 60/310,730, entitled "Alteration of the substrate
specificity of a modular PKS AT domain," which is incorporated
herein by reference. Details of this are provided below in Example
6.
[0082] In another embodiment of the invention, the coding sequence
for the reduction cassette of module 6, which has both DH and KR
domains, is replaced with a coding sequence for a reduction
cassette that has only a KR domain. Details of this are provided
below in Example 7.
[0083] In another embodiment of the invention, inactivation of the
dehydraase domain in module 6 of the geldanamycin PKS gene by
site-specific mutation of the wild-type domain in accord with the
methods of the present invention results in production of
4,5-dihydro-5-hydroxygeldanamycin. Details of this are provided
below in Example 8.
[0084] In another embodiment of the invention, a substantial
portion of the nucleotide sequence in module 6 between the end of
the AT domain is deleted to provide
4,5-dihydro-5-hydroxy-geldanamycin. Details of this are provided
below in Example 9.
[0085] In another embodiment of the invention, the dehydratase
domain of module 1 is replaced or inactivated as described above
for module 6 to provide 15-hydroxy-geldanamycin. Details of this
are provided below in Example 10.
[0086] In another embodiment of the invention, inactivation of the
dehydratase domain in module 1 of the geldanamycin PKS gene by
site-specific mutation of the wild-type domain in accord with the
methods of the present invention results in production of
15-hydroxygeldanamycin. Details of this are provided below in
Example 11.
[0087] It is also possible to express the geldanamycin gene cluster
or mutated versions of the geldanamycin gene cluster prepared
according to the methods of the invention in host cells other than
the native geldanamycin producer. Methods for heterologous
expression of PKS genes and host cells suitable for expression of
these genes and production of polyketides are described, for
example, in U.S. Pat. Nos. 5,843,718 and 5,830,750; PCT
publications WO 01/31035 and WO 01/27306; and U.S. patent
application Ser. Nos. 10/087,451; 60/______, entitled "Process and
Metabolic Strategies for Improved Production of E. coli derived
6-deoxyerythronolide B," by inventors Pfeifer and Khosla (atty
docket no. 30226.00); and 60/396,513, entitled "Metabolic Pathways
for Starter Units in Polyketide Biosynthesis in E. Coli" by
inventors Kealey, Dayem, and Santi; each of which is incorporated
herein by reference.
[0088] Inactivation of dehydratase domains in accord with the
methods of the present invention may also be obtained through
random mutagenesis of the organism that normally produces
geldanamycin. In this instance, spores of the producing organism
can be either treated with a chemical mutagen, for example
1-methyl-3-nitro-1-nitrosoguanidine (MNNG), dimethylsulfate, or the
like, or with mutagenic levels of radiation, for example
ultraviolet radiation. The surviving spores are then allowed to
grow on a suitable medium, and the resulting cultures are analyzed,
for example by LC-mass spectrometry, for the presence of the
desired new geldanamycin analog. Methods for the random mutagenesis
of Streptomyces are described in Kieser et al, "Practical
Streptomyces Genetics," The John Innes Foundation, Norwich (2000),
which is incorporated herein by reference.
[0089] In accordance with the methods of the present invention,
replacement of other acyltransferase domains in the geldanamycin
PKS can be used to generate the respective desmethyl or desmethoxy
analogs, and replacement of other dehydratase domains with inactive
versions can be used to generate the corresponding dihydro-hydroxy
analogs. Such analogs are expected to be more water-soluble, as
they have fewer lipophilic substituents (28-desmethylgeldanamycin)
or have additional hydrophilic substituents
(4,5-dihydro-5-hydroxygeldanamycin or 15-hydroxygeldanamycin).
[0090] By using the geldanamycin analogs produced by genetic
engineering of the geldanamycin PKS in accord with the methods of
the present invention as described above, the afore-mentioned
chemical transformations can be used to convert the analogs into
more water-soluble, more potent, more specific inhibitors of Hsp90.
Such compounds thus may overcome the necessity of using toxic
vehicles such as Cremophore.RTM. in their administration, and show
improved selectivity and reduced side-effects. In one embodiment of
the invention, a geldanamycin analog prepared by genetic
engineering is reacted with an amine as illustrated in Scheme 1
above.
[0091] Ring-forming olefin metathesis of
11-O-allyl-17-allylamino-17-desmethoxy-geldanamycin or similar
analogs generates conformationally constrained benzoquinone
ansamycins structure in accordance with the methods of the present
invention. In one embodiment of the invention, treatment of 17-AAG
with an allylating reagent, such as allyl tert-butyl carbonate and
a palladium catalyst, generates
11-O-allyl-17-allylamino-17-desmethoxy-geldanamycin, as illustrated
in Scheme 4 and exemplified in Example 2.
##STR00011##
[0092] Reaction of related geldanamycin analogs resulting from
displacement of the 17-methoxy group with amines other than
allylamine, e.g., but-3-en-1-ylamine and pent-4-en-1-ylamine,
results in formation of the corresponding
17-butenylamino-11-O-allyl and 17-pentenylamino-11-O-allyl analogs
in accordance with the methods of the present invention. Treatment
of these 11-O-allyl compounds with an olefin metathesis catalyst,
such as benzylidene bis(tricyclohexylphosphine)ruthenium
dichloride, as illustrated in Scheme 5, results in linkage of the
11- and 17-positions through a carbon chain and constraint of the
geldanamycin conformation. This is exemplified below in Example
3.
##STR00012##
[0093] Alteration of the 11-O-- and 17-N-- groups in accord with
the methods of the present invention allows for variation of the
linker chain length. Thus, use of
11-O-allyl-17-allylamino-17-desmethoxygeldanamycin results in a
4-carbon linker, use of
1-O-allyl-17-(but-3-en-1-ylamino)-17-desmethoxygeldanamycin results
in a 5-carbon linker, and use of
11-O-allyl-17-(pent-4-en-1-ylamino)-17-desmethoxygeldanamycin
results in a 6-carbon linker. The carbon-carbon double bond of the
linker can optionally be reduced by reduction, e.g. using diimide,
to provide a saturated linker.
[0094] An alkynyl linker can be prepared according to the methods
of the invention by treating geldanamycin or a geldanamycin analog
with a bifunctional alkyne comprising an amino function at one end
of the linker and a displaceable function, for example a halogen or
sulfonate ester, at the other end. Reaction of geldanamycin or an
analog with this bifunctional linker results first in displacement
of the 17-methoxy group by the amine. Subsequent base treatment
results in alkylation of the 11-hydroxyl as illustrated in Scheme
6.
##STR00013##
[0095] As described above, alteration of the linker lengths can be
achieved through variation in the number of carbon atoms in the
chain.
[0096] In another aspect of the invention, a composition comprising
a benzoquinone ansamycin is used to treat a disease or condition
characterized by undesired cellular proliferation or
hyperproliferation. In one embodiment, the disease is cancer. In
another embodiment, the disease is stenosis or restenosis. In
another embodiment, the disease is psoriasis. In another
embodiment, the disease is a neurodegenerative disease. In
preferred embodiments, the benzoquinone ansamycin is a compound
having formula (I), 17-AAG, or 17-DMAG.
[0097] In another aspect of the invention, a benzoquinone ansamycin
is used in combination therapy with a second agent. In one
embodiment, the second agent is an inhibitor of an Hsp90 client
protein. Suitable Hsp90 client proteins include but are not limited
to those listed in Table 1.
[0098] In one embodiment of the invention, a benzoquinone ansamycin
is used in combination therapy with a protein kinase inhibitor.
Suitable protein kinase inhibitors include but are not limited to
the compounds listed in Table 2.
TABLE-US-00003 TABLE 2 Illustrative list of protein kinase
inhibitors Compound target most advanced indication Quinazolines
and related heterocycles: Iressa (ZD 1839) EGFR non-small cell lung
cancer Tarceva (OSI-774) EGFR ovarian cancer GW2016 EGFR cancer CI
1033 EGFR cancer AZD6474 VEGFR solid tumors
Phenylamino-pyrimidines: Gleevec (STI-571) bcr-abl, others chronic
myelogenous leukemia Pyrazolopyrimidines and pyrrolopyrimidines:
BIBX 1382 EGFR cancer PKI 166 EGFR cancer Indoles and oxindoles:
Semaxanib (SU5416) flk-1/KDR kinase advanced colorectal cancer
SU5402 FGFR cancer Benzylidene malononitriles (tyrphostins):
Tyrphostin 25 EGFR cancer SU101 PDGFR solid tumors Lefunamide
(SU0020) PDGFR solid tumors Flavones: Flavopiridol cdk cancer
B43-genistein LYN leukemia Staurosporines: CEP-701 flt-3 prostate,
pancreatic cancers CEP-2563 trk prostate, other cancers UCN-01
(NSC638850) cancer LY-333531 PKC.beta. diabetic complications
Antibodies, ribozymes: Herceptin Her-2 breast cancer Avastin
antibody VEGFR advanced solid tumors 2c4 antibody Her-2 solid
tumors IMC-1C11 VEGFR metastatic colorectal cancer Cetuximab (C255)
EGFR head &neck squamous cell cancer ABX-EGF EGFR cancer
TheraCIM (H-R3) EGFR metastatic squamous cell carcinoma SMART
anti-VEGF VEGFR relapsed, refractory solid tumors Angiozyme VEGFR
cancer
[0099] The protein kinase inhibitors listed in Table 2 may be
classified according to their chemotypes, including: quinazolines,
particularly 4-anilinoquinazolines such as Iressa (AstraZeneca;
N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quin-
azolinamine) and Tarceva (Roche/Genentech;
N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-Quinazolinamine
monohydrochloride); phenylamino-pyrimidines such as Gleevec
(Novartis;
4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyri-
midinyl]amino]phenyl]benzamide); pyrrolo- and pyrazolopyrimidines
such as BIBX 1382 (Boehringer Ingelheim;
N8-(3-chloro-4-fluorophenyl)-N2-(1-methyl-4-piperidinyl)-pyrimido[5,4-d]p-
yrimidine-2,8-diamine); indoles and oxindoles such as Semaxinib
(Pharmacia;
3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-Indol-2-one);
benzylidene malononitriles; flavones such as flavopiridol (Aventis;
2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidi-
nyl]-4H-1-benzopyran-4-one); staurosporines such as CEP-701
(Cephalon); antibodies such as Herceptin (Genentech); and ribozymes
such as Angiozyme (Ribozyme Pharmaceuticals). Structures of
representative protein kinase inhibitors are given in FIG. 4.
[0100] In another embodiment of the invention, a benzoquinone
ansamycin is used in combination therapy with a microtubule
stabilizing agent, including paclitaxel, epothilone,
discodermolide, and laulimalide. In preferred embodiments, the
benzoquinone ansamycin is a compound having the formula (I),
17-AAG, or 17-DMAG.
[0101] In another aspect, the present invention provides
combination therapy methods for the treatment of diseases or
conditions characterized by undesired cellular proliferation or
hyperproliferation. Combination of two or more drugs in therapy may
result in one of three outcomes: (1) additive, i.e., the effect of
the combination is be equal to the sum of the effects of each drug
when administered alone; (2) synergistic, i.e., the effect of the
combination is greater than the sum of the effects of each drug
when administered alone; or (3) antagonistic, i.e., the effect of
the combination is less than the sum of the effects of each drug
when administered alone. In one embodiment of the present
invention, a subject is first treated with a substantially
sub-toxic dose of a protein kinase inhibitor. After waiting for a
period of time sufficient to allow development of a substantially
efficacious response to the administration of the protein kinase
inhibitor, a synergistic dose of a benzoquinone ansamycin is
administered. Using this dosing schedule, a synergistic rather than
additive effect of the two compounds is achieved. In one embodiment
of the invention, the protein kinase inhibitor is a compound listed
in Table 2, and the benzoquinone ansamycin is a compound having
formula (I), 17-AAG, or 17-DMAG. In another embodiment of the
invention, the protein kinase inhibitor is a drug approved by the
Federal Drug Administration as a stand-alone treatment for cancer,
and the benzoquinone ansamycin is a compound having formula (I),
17-AAG, or 17-DMAG. In one preferred embodiment, the cytotoxic
agent is Iressa and the benzoquinone ansamycin is 17-AAG or
17-DMAG.
[0102] In another embodiment of the invention, a subject is first
treated with a first sub-toxic dose of a protein kinase inhibitor.
After waiting for a period of time sufficient to allow development
of a substantially efficacious response of the protein kinase
inhibitor, a formulation comprising a synergistic dose of a
benzoquinone ansamycin together with a second sub-toxic dose of the
protein kinase inhibitor is administered. In general, the
appropriate period of time sufficient to allow development of a
substantially efficacious response to the protein kinase inhibitor
will depend upon the pharmacokinetics of the protein kinase
inhibitor, and will have been determined during clinical trials of
therapy using the protein kinase inhibitor alone. In one embodiment
of the invention, the period of time sufficient to allow
development of a substantially efficacious response to the protein
kinase inhibitor is between 1 hour and 96 hours. In another
embodiment of the invention, the period of time sufficient to allow
development of a substantially efficacious response to the protein
kinase inhibitor is between 2 hours and 48 hours. In another
embodiment of the invention, the period of time sufficient to allow
development of a substantially efficacious response to the protein
kinase inhibitor is between 4 hours and 24 hours.
[0103] The protein kinase inhibitors are selected from but are not
limited to those listed in Table 2. As demonstrated below in
Example 4 and in FIG. 3A, pretreatment of cultured SKBr3 cells with
the EGFR inhibitor Iressa followed by treatment with 17-AAG results
in synergistic enhancement of the effects of Iressa. In contrast,
the reverse order of administration or simultaneous administration
results in an additive. Similar results were obtained with Iressa
and 17-DMAG, as shown in FIG. 3B. Thus to obtain the optimal
synergistic effect, it is necessary to provide the protein kinase
inhibitor first, wait a period of time sufficient to allow
development of a substantially efficacious response to the protein
kinase inhibitor, and then provide the benzoquinone ansamycin.
[0104] In another embodiment, the subject is first treated with a
sub-toxic dose of a benzoquinone ansamycin. After waiting for a
period of time sufficient to allow development of a substantially
efficacious response to the benzoquinone ansamycin, a synergistic
dose of a microtubule stabilizing agent is administered. Using this
dosing schedule, a synergistic rather than additive effect of the
two compounds is achieved. Unexpectedly in light of Munster et al.
"Modulation of Hsp90 function by ansamycins sensitizes breast
cancer cells to chemotherapy-induced apoptosis in an RB- and
schedule-dependent manner," Clinical Cancer Research (2001) 7:
2228-2236, pretreatment of cultured SKBr3 cells with the
microtubule stabilizing agent paclitaxel followed by treatment with
17-AAG results in an additive effect, as demonstrated below in
Example 4 and in FIG. 5. In contrast, the reverse order of
administration results in a synergistic effect. Illustrative
examples of microtubule stabilizing agents include but are not
limited to paclitaxel, docetaxel, epothilone, discodermolide, and
laulimalide. The benzoquinone ansamycin may be a compound having
formula (I), 17-AAG, or 17-DMAG. In one embodiment of the
invention, the microtubule stabilizing agent is paclitaxel an
epothilone, discodermolide or an analog, or laulimalide or an
analog. In a preferred embodiment of the invention, the microtubule
stabilizing agent is epothilone D.
[0105] In another embodiment of the invention, the combination
therapy the second agent is a drug approved by the Federal Drug
Administration as a stand-alone treatment for cancer, and the
benzoquinone ansamycin is a compound having formula (I) or is
17-AAG or 17-DMAG. Illustrative examples of suitable drugs include
but are not limited to 5-fluorouracil, methotrexate, vinblastine,
cyclophosphamide, mechlorethamine, chlorambucil, Melphalan,
Ifosfamide, bleomycin, mitomycin and doxorubicin.
[0106] In another embodiment, the combination therapy may include
an agent or procedure to mitigate potential side effects from the
combination therapy agents. Diarrhea may be treated with
antidiarrheal agents such as opioids (e.g. codeine, diphenoxylate,
difenoxin, and loeramide), bismuth subsalicylate, and octreotide.
Nausea and vomiting may be treated with antiemetic agents such as
dexamethasone, metoclopramide, diphenyhydramine, lorazepam,
ondansetron, prochlorperazine, thiethylperazine, and dronabinol.
For those compositions that includes polyethoxylated castor oil
such as Cremophor.RTM., pretreatment with corticosteroids such as
dexamethasone and methylprednisolone and/or H1 antagonists such as
diphenylhydramine HCl and/or H2 antagonists may be used to mitigate
anaphylaxis.
[0107] The dose of the second agent when used in combination
therapy with a benzoquinone ansamycin is determined based on the
maximum tolerated dose observed when the second agent is used as
the sole therapeutic agent (the "MTD"). In one embodiment of the
invention, the dose of the second agent when used in combination
therapy with a benzoquinone ansamycin is the MTD. In another
embodiment of the invention, the dose of the second agent when used
in combination therapy with a benzoquinone ansamycin is between 1%
of the MTD and the MTD. In another embodiment of the invention, the
dose of the second agent when used in combination therapy with a
benzoquinone ansamycin is between 5% of the MTD and the MTD. In
another embodiment of the invention, the dose of the second agent
when used in combination therapy with a benzoquinone ansamycin is
between 5% of the MTD and 75% of the MTD. In another embodiment of
the invention, the dose of the second agent when used in
combination therapy with a benzoquinone ansamycin is between 25% of
the MTD and 75% of the MTD.
[0108] Use of the benzoquinone ansamycin allows for use of a lower
therapeutic dose of the second agent, thus significantly widening
the therapeutic window for treatment. In one embodiment, the
therapeutic dose of the second agent is lowered by at least 10%. In
another embodiment, the therapeutic dose of the second agent is
lowered from 10 to 20%. In another embodiment, the therapeutic dose
of the second agent is lowered from 20 to 50%. In another
embodiment, the therapeutic dose of the second agent is lowered
from 50 to 200%. In another embodiment, the therapeutic dose of the
second agent is lowered from 100 to 1000%.
[0109] The MTD for a compound is determined using methods and
materials known in the medical and pharmacological arts, for
example through dose-escalation experiments. One or more patients
is first treated with a low dose of the compound, typically 10% of
the dose anticipated to be therapeutic based on results of in vitro
cell culture experiments. The patients are observed for a period of
time to determine the occurrence of toxicity. Toxicity is typically
evidenced as the observation of one or more of the following
symptoms: vomiting, diarrhea, peripheral neuropathy, ataxia,
neutropenia, or elevation of liver enzymes. If no toxicity is
observed, the dose is increased 2-fold, and the patients are again
observed for evidence of toxicity. This cycle is repeated until a
dose producing evidence of toxicity is reached. The dose
immediately preceding the onset of unacceptable toxicity is taken
as the MTD.
[0110] The synergistic dose of the benzoquinone ansamycin used in
combination therapy is determined based on the maximum tolerated
dose observed when the benzoquinone ansamycin in used as the sole
therapeutic agent. Clinical trials have determined an MTD for
17-AAG of 40 mg/m.sup.2. In one embodiment of the invention, the
dose of the benzoquinone ansamycin when used in combination therapy
is the MTD. In another embodiment of the invention, the dose of the
benzoquinone ansamycin when used in combination therapy is between
1% of the MTD and the MTD. In another embodiment of the invention,
the dose of the benzoquinone ansamycin when used in combination
therapy is between 5% of the MTD and the MTD. In another embodiment
of the invention, the dose of the benzoquinone ansamycin when used
in combination therapy is between 5% of the MTD and 75% of the MTD.
In another embodiment of the invention, the dose of the
benzoquinone ansamycin when used in combination therapy is between
25% of the MTD and 75% of the MTD.
[0111] The dosages of the benzoquinone ansamycin and the Hsp90
client protein inhibitor when used in combination therapy may
require further optimization depending upon the compounds being
used, the disease or condition being treated, and the individual
medical condition of the patient. Relevant factors include the
activity of the specific compound employed; the age, body weight,
general health, sex, and diet of the subject; the time and route of
administration and the rate of excretion of the drug; and the
severity of the condition being treated.
[0112] The present invention provides compositions of matter that
are formulations of one or more active drugs and a pharmaceutically
acceptable carrier. In one embodiment, the formulation comprises a
novel benzoquinone ansamycin analog of the invention. In another
embodiment, the formulation comprises a novel benzoquinone
ansamycin analog of the invention as a mixture with an Hsp90 client
protein inhibitor for use in combination therapy in accord with the
methods of the present invention. In both of these embodiments, the
active compounds may be in their free form or where appropriate as
pharmaceutically acceptable derivatives such as prodrugs, esters,
or salts.
[0113] The composition may be in any suitable form such as solid,
semisolid, or liquid form. See Pharmaceutical Dosage Forms and Drug
Delivery Systems, 5th edition, Lippicott Williams & Wilkins
(1991), incorporated herein by reference. In general, the
pharmaceutical preparation will contain one or more of the
compounds of the invention as an active ingredient in admixture
with an organic or inorganic carrier or excipient suitable for
external, enteral, or parenteral application. The active ingredient
may be compounded, for example, with the usual non-toxic,
pharmaceutically acceptable carriers for tablets, pellets,
capsules, suppositories, pessaries, solutions, emulsions,
suspensions, and any other form suitable for use. The carriers that
can be used include water, glucose, lactose, gum acacia, gelatin,
mannitol, starch paste, magnesium trisilicate, talc, corn starch,
keratin, colloidal silica, potato starch, urea, and other carriers
suitable for use in manufacturing preparations, in solid,
semi-solid, or liquified form. In addition, auxiliary stabilizing,
thickening, and coloring agents and perfumes may be used.
[0114] In one embodiment, the compositions containing a compound
useful in the methods of the invention are Cremophore.RTM.-free.
Cremophore.RTM. (BASF Aktiengesellschaft) is a polyethoxylated
castor oil which is typically used as a surfactant in formulating
low soluble drugs. However, because Cremophore.RTM. can case
allergic reactions in a subject, compositions that minimize or
eliminate Cremophore.RTM. are preferred.
[0115] Where applicable, the compounds useful in the methods of the
invention may be formulated as microcapsules and nanoparticles.
General protocols are described for example, by Microcapsules and
Nanoparticles in Medicine and Pharmacy by Max Donbrow, ed., CRC
Press (1992) and by U.S. Pat. Nos. 5,510,118; 5,534,270; and
5,662,883 which are all incorporated herein by reference. By
increasing the ratio of surface area to volume, these formulations
allow for the oral delivery of compounds that would not otherwise
be amenable to oral delivery.
[0116] The compounds useful in the methods of the invention may
also be formulated using other methods that have been previously
used for low solubility drugs. For example, the compounds may form
emulsions with vitamin E or a PEGylated derivative thereof as
described by PCT publications WO 98/30205 and WO 00/71163, each of
which is incorporated herein by reference. Typically, the compound
useful in the methods of the invention is dissolved in an aqueous
solution containing ethanol (preferably less than 1% w/v). Vitamin
E or a PEGylated-vitamin E is added. The ethanol is then removed to
form a pre-emulsion that can be formulated for intravenous or oral
routes of administration. Another method involves encapsulating the
compounds useful in the methods of the invention in liposomes.
Methods for forming liposomes as drug delivery vehicles are well
known in the art. Suitable protocols include those described by
U.S. Pat. Nos. 5,683,715; 5,415,869, and 5,424,073 which are
incorporated herein by reference relating to another relatively low
solubility cancer drug paclitaxel and by PCT Publication WO
01/10412 which is incorporated herein by reference relating to
epothilone B. Of the various lipids that may be used, particularly
preferred lipids for making encapsulated liposomes include
phosphatidylcholine and polyethyleneglycol-derivitized distearyl
phosphatidyl-ethanolamine.
[0117] Yet another method involves formulating the compounds useful
in the methods of the invention using polymers such as polymers
such as biopolymers or biocompatible (synthetic or naturally
occurring) polymers. Biocompatible polymers can be categorized as
biodegradable and non-biodegradable. Biodegradable polymers degrade
in vivo as a function of chemical composition, method of
manufacture, and implant structure. Illustrative examples of
synthetic polymers include polyanhydrides, polyhydroxyacids such as
polylactic acid, polyglycolic acids and copolymers thereof,
polyesters polyamides polyorthoesters and some polyphosphazenes.
Illustrative examples of naturally occurring polymers include
proteins and polysaccharides such as collagen, hyaluronic acid,
albumin, and gelatin.
[0118] Another method involves conjugating the compounds useful in
the methods of the invention to a polymer that enhances aqueous
solubility. Examples of suitable polymers include polyethylene
glycol, poly-(d-glutamic acid), poly-(l-glutamic acid),
poly-(l-glutamic acid), poly-(d-aspartic acid), poly-(l-aspartic
acid), poly-(l-aspartic acid) and copolymers thereof. Polyglutamic
acids having molecular weights between about 5,000 to about 100,000
are preferred, with molecular weights between about 20,000 and
80,000 being more preferred and with molecular weights between
about 30,000 and 60,000 being most preferred. The polymer is
conjugated via an ester linkage to one or more hydroxyls of an
inventive geldanamycin using a protocol as essentially described by
U.S. Pat. No. 5,977,163 which is incorporated herein by
reference.
[0119] In another method, the compounds useful in the methods of
the invention are conjugated to a monoclonal antibody. This method
allows the targeting of the inventive compounds to specific
targets. General protocols for the design and use of conjugated
antibodies are described in Monoclonal Antibody-Based Therapy of
Cancer by Michael L. Grossbard, ed. (1998), which is incorporated
herein by reference.
[0120] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the subject treated and the particular mode of
administration. For example, a formulation for intravenous use
comprises an amount of the inventive compound ranging from about 1
mg/mL to about 25 mg/mL, preferably from about 5 mg/mL to 15 mg/mL,
and more preferably about 10 mg/mL. Intravenous formulations are
typically diluted between about 2 fold and about 30 fold with
normal saline or 5% dextrose solution prior to use.
[0121] In one aspect of the present invention, the compounds useful
in the methods of the invention are used to treat cancer. In one
embodiment, the compounds of the present invention are used to
treat cancers of the head and neck which include but are not
limited to tumors of the nasal cavity, paranasal sinuses,
nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary
glands, and paragangliomas. In another embodiment, the compounds of
the present invention are used to treat cancers of the liver and
biliary tree, particularly hepatocellular carcinoma. In another
embodiment, the compounds of the present invention are used to
treat intestinal cancers, particularly colorectal cancer. In
another embodiment, the compounds of the present invention are used
to treat ovarian cancer. In another embodiment, the compounds of
the present invention are used to treat small cell and non-small
cell lung cancer. In another embodiment, the compounds of the
present invention are used to treat breast cancer. In another
embodiment, the compounds of the present invention are used to
treat sarcomas, including fibrosarcoma, malignant fibrous
histiocytoma, embryonal rhabdomysocarcoma, leiomysosarcoma,
neuro-fibrosarcoma, osteosarcoma, synovial sarcoma, liposarcoma,
and alveolar soft part sarcoma. In another embodiment, the
compounds of the present invention are used to treat neoplasms of
the central nervous systems, particularly brain cancer. In another
embodiment, the compounds of the present invention are used to
treat lymphomas which include Hodgkin's lymphoma,
lymphoplasmacytoid lymphoma, follicular lymphoma, mucosa-associated
lymphoid tissue lymphoma, mantle cell lymphoma, B-lineage large
cell lymphoma, Burkitt's lymphoma, and T-cell anaplastic large cell
lymphoma.
[0122] In another embodiment, the compounds and compositions useful
in the methods of the invention are used at sub-cytotoxic levels in
combination with other agents in order to achieve highly selective
activity in the treatment of non-cancerous diseases. In this
embodiment, the compounds useful in the methods of the invention
are used to reduce the cellular levels of Hsp90 client proteins,
which are then effectively inhibited by the second agent. Binding
of the client proteins to Hsp90 stabilizes the client proteins and
maintains them in a soluble, inactive form ready to respond to
activating stimuli. Binding of a benzoquinone ansamycin analog to
Hsp90 results in targeting of the client protein to the proteasome,
and subsequent degradation. For systems such as the steroid
receptor, however, Hsp90 forms an integral part of the functional
receptor complex along with several other proteins such as Hsp70,
Hsp40, p23, hip, Hsp56, and immunophilins. Hsp90 appears to
regulate the activity of the steroid receptor by maintaining the
receptor in a high-affinity hormone-binding conformation. Binding
of geldanamycin to Hsp90 appears to result in dissociation of p23
from the complex and reduce the level of hormone binding to the
receptor (Fliss et al. (2000) "Control of estrogen receptor ligand
binding by Hsp90," J. Steroid Biochem. Mol. Biol. 75: 223-30;
Kimmins and MacRae (2000), "Maturation of steroid receptors: an
example of functional cooperation among molecular chaperones and
their associated proteins," Cell Stress Chaperones 5: 76-86.
[0123] Thus, Hsp90 inhibitors such as geldanamycin, geldanamycin
analogs, radicicol, and the like can be used in accord with the
methods of the present invention to alter the function of hormone
receptors, making it easier to inhibit the associated signal
pathways using low levels of a second drug which targets the
proteins involved in those signaling pathways. Such a combination
therapy can be useful to reduce non-specific toxicity associated
with therapy by reducing the levels of the drugs required.
[0124] In another embodiment, the compounds useful in the methods
of the invention are used to treat non-cancerous diseases or
conditions characterized by undesired cellular hyperproliferation,
including neurodegenerative diseases, psoriasis, stenosis, and
restenosis.
[0125] In another embodiment, the compounds useful in the methods
of the invention are used in combination with other agents as
described above to treat non-cancerous diseases or conditions
characterized by undesired cellular hyperproliferation, including
neuro-degenerative diseases, psoriasis, stenosis, and restenosis.
Specific examples of non-cancerous diseases treatable by this
combination therapy include neurodegenerative diseases, such as
Alzheimer's, Parkinson's, Huntington's and the like. There is
evidence that the immunophilin FKBP-52 is involved as a client
protein for Hsp90 in the formation of various steroid receptor
complexes and plays a role in the regeneration of damaged neurons
(Gold et al., "Immunophilin FK506-Binding Protein 52 (Not
FK506-Binding Protein 12) Mediates the Neurotrophic Action of
FK506," 1999, J. Pharmacology & Exp. Ther. 289: 1202-1210). The
combination of geldanamycin, a geldanamycin analog, radicicol, or
the like with a second agent which binds to and/or inhibits FKBP-52
in accord with the methods of the present invention can thus be
used to treat neurodegenerative diseases.
[0126] Further examples of non-cancerous diseases treatable by the
combination therapy of the present invention include non-cancerous
diseases characterized by cellular hyperproliferation, such as
psoriasis, stenosis, and restenosis. Cell proliferation is
regulated by protein tyrosine kinases, many of which are known to
be client proteins for Hsp90. Psoriasis is thought to involve the
epidermal growth factor receptor (EGFR), a protein tyrosine kinase,
and inhibitors of EGFR have been proposed as treatments for
psoriasis (Ben-Bassat & Klein, "Inhibitors of Tyrosine Kinases
in the Treatment of Psoriasis," (2000), Curr. Pharm. Des. 6:
933-942). Geldanamycin and herbimycin have been shown to block
maturation of EGFR (Sakagami et al., "Benzoquinoid ansamycins
(herbimycin A and geldanamycin) interfere with the maturation of
growth factor receptor tyrosine kinases," (1999) Cell Stress
Chaperones 4: 19-28). The combination of geldanamycin or a
geldanamycin analog with an inhibitor of EGFR in accord with the
methods of the present invention can thus be used to treat
psoriasis.
[0127] The compounds useful in the methods of the invention may
also be used to treat stenosis and restenosis, particularly
associated with in vivo devices such as stents. In one embodiment,
the compounds useful in the methods of the invention are used to
coat stents and other surgically-implantable devices. In another
embodiment, the compounds useful in the methods of the invention
are used in combination with other agents as described above to
coat stents, catheters, prostheses, and other in vivo devices.
[0128] A detailed description of the invention having been provided
above, the following examples are given for the purpose of
illustrating the present invention and shall not be construed as
being a limitation on the scope of the invention or claims.
EXAMPLE 1
Preparation of 17-alkylamino-17-desmethoxygeldanamycins
[0129] The general procedure for preparing
17-alkylamino-17-desmethoxygeldanamycins is as follows. The
geldanamycin analog (25 mmol) is suspended in 350 mL of anhydrous
CH.sub.2Cl.sub.2 under inert atmosphere. A solution of the
alkylamine (50 mmol) in 10 mL of CH.sub.2Cl.sub.2 is added
dropwise. After 1 hour, the mixture is evaporated to dryness and
the residue is dissolved in 50 mL of chloroform and precipitated by
addition of 600 mL of hexane. Filtration and vacuum drying yields
the product. The products are characterized by NMR and LC/MS.
Compounds prepared according to this method include: [0130]
17-allylamino-17-desmethoxygeldanamycin; [0131]
17-(2-(dimethylamino)ethyl)amino-17-desmethoxygeldanamycin; [0132]
17-ethylamino-17-desmethoxygeldanamycin; [0133]
17-propylamino-17-desmethoxygeldanamycin; [0134]
17-butylamino-17-desmethoxygeldanamycin; [0135]
17-(cyclopropyl)methylamino-17-desmethoxygeldanamycin; [0136]
17-cyclobutylamino-17-desmethoxygeldanamycin; [0137]
17-(2-phenylcyclopropyl)amino-17-desmethoxygeldanamycin; [0138]
17-(2-fluoroethyl)amino-17-desmethoxygeldanamycin; [0139]
17-(2,2-difluoroethyl)amino-17-desmethoxygeldanamycin; [0140]
17-azetidinyl-17-desmethoxygeldanamycin; [0141]
17-amino-17-desmethoxygeldanamycin; [0142]
17-(3-(dimethylamino)propylamino-17-desmethoxygeldanamycin; [0143]
17-(4-(dimethylamino)butylamino-17-desmethoxygeldanamycin; [0144]
17-(3-dimethylamino)-2-propylamino-17-desmethoxygeldanamycin;
[0145]
17-(2-dimethylamino)-1-propylamino-17-desmethoxygeldanamycin;
[0146]
17-(N-ethylpyrrolidin-2-yl)methylamino-17-desmethoxygeldanamycin;
[0147] 17-(1-pyrazinyl)ethylamino-17-desmethoxygeldanamycin; [0148]
17-(2-bis(2-hydroxyethyl)amino)ethylamino-17-desmethoxygeldanamycin;
[0149]
17-(N-methylpyrrolidin-2-yl)ethylamino-17-desmethoxygeldanamycin;
[0150] 17-(3-(diethylamino)propylamino-17-desmethoxygeldanamycin;
[0151] 17-(3-(4-morpholino)propylamino-17-desmethoxygeldanamycin;
[0152] 17-(4-imidazolyl)ethylamino-17-desmethoxygeldanamycin;
[0153]
17-(1-methyl-4-imidazolyl)ethylamino-17-desmethoxygeldanamycin;
[0154]
17-(1-methyl-5-imidazolyl)ethylamino-17-desmethoxygeldanamycin;
[0155] 17-(4-pyridyl)ethylamino-17-desmethoxygeldanamycin; [0156]
17-(2-hydroxyethyl)amino-17-desmethoxygeldanamycin; [0157]
17-(1-hydroxymethyl)propylamino-17-desmethoxygeldanamycin; [0158]
17-(2-(hydroxymethyl)cyclohexyl)amino-17-desmethoxygeldanamycin;
[0159] 17-(2-dioxolan-1-yl)ethylamino-17-desmethoxygeldanamycin;
[0160] 17-(3,3-dimethoxypropyl)amino-17-desmethoxygeldanamycin;
[0161]
17-(2-tert-butoxycarbonylamino)ethylamino-17-desmethoxygeldanamycin;
and [0162] 17-(N-methyl
2-tert-butoxycarbonylamino)ethylamino-17-desmethoxygeldanamycin.
EXAMPLE 2
Preparation of
11-O-allyl-17-alkylamino-17-desmethoxygeldanamycins
[0163] The general procedure for preparing
11-O-allyl-17-alkylamino-17-desmethoxy-geldanamycins is as follows.
The 17-alkylamino-17-desmethoxy-geldanamycin analog (10 mmol),
allyl tert-butyl carbonate (14 mmol), palladium acetate (0.1 mmol),
and triphenylphosphine (0.67 mmol) are dissolved in 60 mL of
freshly distilled, air-free tetrahydrofuran under inert atmosphere.
The mixture is heated at reflux for 16 hours, and is monitored by
thin-layer chromatography. Upon completion, the solvents are
removed in vacuo and the product isolated by silica gel
chromatography.
EXAMPLE 3
Preparation of 11,17-linked-17-amino-17-desmethoxygeldanamycins
[0164] The general procedure for preparing
11,17-linked-17-amino-17-desmethoxy-geldanamycins is as follows.
The 111-O-allyl-17-alkylaminogeldanamycin analog (10 mmol), and
benzylidene-bis(tricyclohexylphosphine)ruthenium dichloride (5
mmol) are dissolved in 250 mL of anhydrous CH.sub.2Cl.sub.2 under
inert atmosphere, and the reaction is monitored by thin-layer
chromatography. After 24 hours, the reaction is concentrated and
the residue is purified by silica gel chromatography.
EXAMPLE 4
Interaction of Geldanamycin Analogs and Cytotoxic Agents in SKBr3
Cells
[0165] SKBr3 and H358 cell lines were obtained from American Type
Culture Collection (Manassas, Va.). Cells were maintained in
McCoy'5A medium with 10% fetal bovine serum. 17-AAG and 17-DMAG
were dissolved in DMSO at 10 mM and stored at -20.degree. C.
[0166] Cells were seeded in duplicate in opaque-walled 96-well
microtiter plates at 4000 cells per well and allowed to attach
overnight. Serial dilutions of each drug were added, and the cells
were incubated for 72 hours. The IC.sub.50 was determined using the
CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison,
Wis.), which correlates with the number of live cells.
[0167] For the drug combination assay, cells were seeded in
duplicate in 96-well plates (4000 cells/well). After an overnight
incubation, cells were treated with drug alone or in combination.
Based on the IC.sub.50 values of each individual drug, combined
drug treatment was designed at constant ratios of two drugs, i.e.,
equivalent to the ratio of their IC.sub.50. Three different
treatment schedules were used. The first treatment schedule used
simultaneous exposure to both 17-AAG and the second cytotoxic agent
for 72 hours. In the second schedule, the cells were exposed to 24
hours of 17-AAG or 17-DMAG. The second cytotoxic agent was then
added to the cells and incubated for 48 hours. In the third
treatment schedule, cells were exposed to the second cytotoxic
agent alone for 24 hours followed by addition of 17-AAG or 17-DMAG
for 48 hours. Cell viability was determined by luminescent assay
(Promega). Combination analysis was performed using Calcusyn
software (Biosoft, Cambridge, UK). The "combination index" (CI)
refers to a measure of the additivity of the effects of two drugs
administered in combination, and was calculated according to the
formula:
CI=[D].sub.1/[D.sub.x].sub.1+[D].sub.2/[D.sub.x].sub.2
wherein [D]1 and [D]2 represent the concentrations of the first and
second drug, respectively, that in combination provide a response
of x % in the assay, and [Dx]1 and [Dx]2 represent the
concentrations of the first and second drug, respectively, that
when used alone provide a response of x % in the assay. According
to this formula, a CI=1 indicates a simple additive effect of the
two drugs in combination, whereas a CI<1 indicates synergism and
a CI>1 indicates antagonism between the two drugs.
[0168] Addition of 17-AAG or 17-DMAG to cells after exposure to
Iressa synergistically increased the cytotoxicity of Iressa (FIG.
3). An additive effect was detected when cells were exposed to
17-AAG or 17-DMAG before Iressa. Exposure of cells to 17-AAG or
17-DMAG and Iressa simultaneously also produced an additive effect.
Thus the optimal synergistic effect is only observed when there is
prior exposure of the cells to the protein kinase inhibitor,
followed by exposure to the benzoquinone ansamycin after waiting
for a period of time to allow development of efficacy of the
protein kinase inhibitor.
[0169] In contrast, addition of 17-AAG or 17-DMAG to cells after
exposure to paclitaxel additively increased the cytotoxicity of
paclitaxel (FIG. 5). A synergistic effect was observed when cells
were exposed to paclitaxel first, and were later treated with
17-AAG or 17-DMAG. In this case, optimal synergism is only observed
when the cells are first exposed to the benzoquinone ansamycin,
followed by exposure to the microtubule stabilizing agent after
waiting for a period of time to allow development of efficacy of
the benzoquinone ansamycin.
EXAMPLE 5
Production of 28-desmethylgeldanamycin by AT Domain
Substitution
[0170] Substitution of the acyltransferase domain in module 1 of
the geldanamycin PKS gene with an AT domain specific for
malonyl-CoA instead of 2-methylmalonyl-CoA results in formation of
28-desmethyl-geldanamycin. The domain substitution is created by
introducing a malonyl-CoA specific acyltransferase domain from a
heterologous PKS gene, for example from the rapamycin, tylosin, or
FK520 PKS genes or the like, into the geldanamycin PKS locus by
homologous recombination into a geldanamycin producing strain,
aided by a selectable antibiotic resistance gene, then isolating
the recombinants resulting from double crossover events in which
the wild-type acyltransferase domain is replaced with one specific
for malonyl-CoA. The AT domain of module 1 is encoded by
nucleotides 1626 through 2670, approximately, of SEQ ID NO:1. This
sequence information together with the methods described in U.S.
Pat. Nos. 6,399,789; 6,403,775; and 5,962,290 allows one skilled in
the art to construct recombination vectors that result in
replacement of the native AT domain of module 1 with an AT domain
having a specificity for malonyl-CoA. Suitable examples of AT
domains with specificity for malonyl-CoA may be found in the
rapamycin PKS genes (modules 2, 5, 8, 9, 11, 12, and 14), as
described in U.S. Pat. No. 6,399,789, as well as the tylosin PKS
genes (modules 3 and 7) as described in U.S. Pat. No. 5,876,991;
the spiramycin genes (modules 1-3 and 7), as described in U.S. Pat.
No. 5,945,320; the FK520 genes (modules 3 and 10), as described in
WO 00/20601; the pikromycin genes (module 2) as described in WO
99/61599; the narbomycin genes (module 2), as described in U.S.
Pat. No. 6,303,767; the avermectin genes (module 2), and others.
Each of the above documents is incorporated herein by reference.
Fermentation of a host cell comprising the resulting hybrid PKS
together with the remaining geldanamycin biosynthetic genes under
conditions wherein the native strain produces geldanamycin,
followed by extraction of the broth and purification provides
28-desmethyl-geldanamycin.
EXAMPLE 6
Production of 28-desmethylgeldanamycin by Site-Directed
Mutagenesis
[0171] The acyltransferase domain in module 1 of the geldanamycin
PKS gene is mutagenized according to the methods described in
Reeves et al., "Alteration of the substrate specificity of a
modular polyketide synthase acyltranserase domain through
site-directed mutagenesis," Biochemistry 2001, 40: 15464-15470, and
in U.S. patent application Ser. No. 60/310,730, entitled
"Alteration of the substrate specificity of a modular PKS AT
domain," which is incorporated herein by reference. The amino acid
sequence Tyr-Ala-Ser-His (SEQ ID NO: 3), encoded by nucleotide
sequence TAC-GCC-TCC-CAC (SEQ ID NO: 4) at positions 2194 to 2205
in SEQ ID NO:1, is mutagenized using methods known to one skilled
in the art to generate the mutant amino acid sequence
His-Ala-Phe-His (SEQ ID NO: 5), for example by mutagenesis of the
nucleotide sequence to CAC-GCC-TTC-CAC (SEQ ID NO: 6) as described
in the Reeves et al. reference cited above. Fermentation of a host
cell comprising the resulting mutagenized PKS together with the
remaining geldanamycin biosynthetic genes under conditions wherein
the native strain produces geldanamycin, followed by extraction of
the broth and purification provides 28-desmethyl-geldanamycin.
EXAMPLE 7
Production of 4,5-dihydro-5-hydroxygeldanamycin by Reductive Domain
Swap
[0172] The coding sequence for the reduction cassette of module 6,
which has both DH and KR domains, is replaced with a coding
sequence for a reduction cassette that has only a KR domain. The
reduction cassette is contained in the sequence between the end of
the AT domain, at approximately nucleotide position 2805 of SEQ ID
NO:2, and the beginning of the ACP domain, at approximately
nucleotide position 6028 of SEQ ID NO:2. This sequence information
together with the methods described in U.S. Pat. Nos. 6,399,789;
6,403,775; and 5,962,290 allows one skilled in the art to construct
recombination vectors that result in replacement of the native
reduction cassette of module 6 with a cassette encoding only a KR
domain. Suitable examples of cassettes encoding only a KR domain
may be found in the erythromycin and rapamycin PKS genes, as
described in U.S. Pat. No. 6,399,789. Fermentation of a host cell
comprising the resulting hybrid PKS together with the remaining
geldanamycin biosynthetic genes under conditions wherein the native
strain produces geldanamycin, followed by extraction of the broth
and purification provides 4,5-dihydro-5-hydroxy-geldanamycin.
EXAMPLE 8
Production of 4,5-dihydro-5-hydroxygeldanamycin by Site-Directed
Mutagenesis
[0173] Inactivation of the dehydratase domain in module 6 of the
geldanamycin PKS gene by site-specific mutation of the wild-type
domain results in production of 4,5-dihydro-5-hydroxygeldanamycin.
The DH domain of module 6 is encoded by nucleotides 2805 to 4276,
approximately, of SEQ ID NO:2. Two particular sequences may be
targeted for mutational inactivation of the DH domain. In one
embodiment, the DNA sequence encoding the DH peptide motif
His-Val-Ile-Ser-Gly-Ala-Val-Leu-Val-Pro (SEQ ID NO: 7), nucleotides
2956 through 2985 of SEQ ID NO:2, is mutated so as to produce a
peptide having an amino acid other than histidine at the first
position. The CAC codon encoding histidine is mutated, for example
to CAA or CAG to encode a glutamine, as illustrated in SEQ ID NO:
8. Fermentation of a host cell comprising the resulting mutagenized
PKS together with the remaining geldanamycin biosynthetic genes
under conditions wherein the native strain produces geldanamycin,
followed by extraction of the broth and purification provides
4,5-dihydro-5-hydroxy-geldanamycin.
EXAMPLE 9
Production of 4,5-dihydro-5-hydroxygeldanamycin by Deletion
[0174] A portion of the nucleotide sequence in module 6 between the
end of the AT domain (approximately nucleotide 2805 of SEQ ID NO:2)
and the start of the KR domain (approximately nucleotide 5212 of
SEQ ID NO:2) is deleted to provide a modified PKS for production of
4,5-dihydro-5-hydroxy-geldanamycin. The nucleotide sequence between
2805 and 3270, approximately, of SEQ ID NO:2 is deleted so as to
leave a linker region between the AT and KR domains of
approximately 465 amino acids. Fermentation of a host cell
comprising the resulting modified PKS together with the remaining
geldanamycin biosynthetic genes under conditions wherein the native
strain produces geldanamycin, followed by extraction of the broth
and purification provides 4,5-dihydro-5-hydroxy-geldanamycin.
EXAMPLE 10
Production of 15-hydroxygeldanamycin by Reductive Domain Swap
[0175] The reduction cassette in module 1 is encoded by the
sequence between the end of the AT domain, at approximately
nucleotide position 2670 of SEQ ID NO:1, and the beginning of the
ACP domain, at approximately nucleotide position 5895 of SEQ ID
NO:1. This sequence information together with the methods described
in U.S. Pat. Nos. 6,399,789; 6,403,775; and 5,962,290 allows one
skilled in the art to construct recombination vectors that result
in replacement of the native reduction cassette of module 1 with a
cassette encoding only a KR domain. Suitable examples of cassettes
encoding only a KR domain may be found in the erythromycin and
rapamycin PKS genes, as described in U.S. Pat. No. 6,399,789.
Fermentation of a host cell comprising the resulting hybrid PKS
together with the remaining geldanamycin biosynthetic genes under
conditions wherein the native strain produces geldanamycin,
followed by extraction of the broth and purification provides
15-hydroxy-geldanamycin.
EXAMPLE 11
Production of 15-hydroxygeldanamycin by Site-Directed
Mutagenesis
[0176] Inactivation of the dehydratase domain in module 1 of the
geldanamycin PKS gene by site-specific mutation of the wild-type
domain results in production of 15-hydroxygeldanamycin. The DH
domain of module 1 is encoded by nucleotides 2670 to 4140,
approximately, of SEQ ID NO:1. Two particular sequences may be
targeted for mutational inactivation of the DH domain. In one
embodiment, the DNA sequence encoding the DH peptide motif
His-Ala-Val-Ser-Gly-Thr-Val-Leu-Leu-Pro (SEQ ID NO: 9), nucleotides
2821 through 2850 of SEQ ID NO:1, is mutated so as to produce a
peptide having an amino acid other than histidine at the first
position. The CAC codon encoding histidine is mutated, for example
to CAA or CAG to encode a glutamine as illustrated in SEQ ID NO:
10. Fermentation of a host cell comprising the resulting
mutagenized PKS together with the remaining geldanamycin
biosynthetic genes under conditions wherein the native strain
produces geldanamycin, followed by extraction of the broth and
purification provides 15-hydroxy-geldanamycin.
EXAMPLE 12
8,9-epoxy-8,9-dihydrogeldanamycin
[0177] To a solution of geldanamycin (120 mg, 0.21 mmol) in
dichloromethane (5 mL) was added 85% 3-chloroperoxybenzoic acid (93
mg, 0.42 mmol). After the mixture was stirred at room temperature
overnight, TLC showed the reaction to be complete. The reaction
mixture was diluted with ethyl acetate and washed subsequently with
aqueous sodium sulfite, aqueous bicarbonate, and brine. The organic
solution was dried over anhydrous sodium sulfate, filtered, and
evaporated to dryness. The crude product was purified by flash
chromatography on silica gel, giving 30 mg of
8,9-epoxy-8,9-dihydrogeldanamycin as a yellow solid. The compound
was characterized by NMR and MS spectrometry.
EXAMPLE 13
8,9-epoxy-17-[2-(dimethylamino)ethylamino]-8,9-dihydro-17-demethyoxygeldan-
amycin
[0178] To a solution of 8,9-epoxy-8,9-dihydrogeldanamycin (20 mg,
0.035 mmol) in 1,2-dichloroethane (2 mL) was added
N,N-dimethylethylenediamine (10 .mu.L, 0.09 mmol). The mixture was
stirred at room temperature overnight. The reaction was worked up
by diluting with ethyl acetate and washing sequentially with
aqueous bicarbonate and brine. The organic solution was dried over
anhydrous sodium sulfate, filtered, and evaporated to dryness. The
crude product was purified by HPLC on a C-18 column, giving 10 mg
of
8,9-epoxy-17-[2-(dimethylamino)ethylamino]-8,9-dihydro-17-demethyoxygelda-
namycin as a purple solid. The compound was characterized by NMR
and MS spectrometry.
EXAMPLE 14
17-[2-(dimethylamino)ethylamino]-17-demethyoxygeldanamycin
[0179] To a solution of geldanamycin (60 mg, 0.1 mmol) in
1,2-dichloroethane (4 mL) was added N,N-dimethylethylenediamine (22
.mu.L, 0.2 mmol). The mixture was stirred at room temperature
overnight. The reaction was diluted with ethyl acetate and washed
sequentially with aqueous bicarbonate and brine. The organic
solution was dried over anhydrous sodium sulfate, filtered, and
evaporated to dryness. The crude product was purified by flash
chromatography on silica gel, giving 59 mg of
17-[2-(dimethylamino)-ethylamino]-17-demethyoxygeldanamycin as a
purple solid. The compound was characterized by NMR and MS
spectrometry.
EXAMPLE 15
17-[2-(dimethylamino)ethylamino]-11-oxo-17-demethyoxygeldanamycin
[0180] To a solution of
17-[2-(dimethylamino)ethylamino]-17-demethyoxygeldanamycin (50 mg,
0.08 mmol) in chloroform (5 ml) was added the Dess-Martin
periodinane (208 mg, 0.49 mmol). The mixture was heated to reflux
for one hour. The reaction mixture was diluted in ethyl acetate and
washed subsequently with aqueous sodium thiosulfate, aqueous
bicarbonate, and brine. The organic solution was dried over
anhydrous sodium sulfate, filtered, and evaporated to dryness. The
crude product was purified by HPLC on a C-18 column, giving 26 mg
of
17-[2-(dimethylamino)ethylamino]-11-oxo-17-demethyoxygeldanamyci- n
as a purple solid. The compound was characterized by NMR and MS
spectrometry.
EXAMPLE 16
17-[2-(dimethylamino)ethylamino]-11-oxo-17-demethyoxygeldanamycin-11-oxime
[0181] To a solution of
17-[2-(dimethylamino)ethylamino]-11-oxo-17-demethyoxy-geldanamycin
(30 mg, 0.049 mmol) in ethanol (4 mL) was added triethylamine (60
.mu.L, 0.43 mmol) and hydroxylamine hydrochloride (30 mg, 0.4
mmol). After the mixture was stirred at room temperature for 3 h,
TLC showed reaction complete. After ethanol was evaporated, the
residue was dissolved in chloroform and water. The organic layer
was dried over anhydrous sodium sulfate, filtered, and evaporated
to dryness. The crude product was purified by flash chromatography
on silica gel, giving 10 mg of
17-[2-(dimethylamino)ethylamino]-11-oxo-17-demethyoxygeldanamycin-11-oxim-
e as a purple solid. The compound was characterized by NMR and MS
spectrometry.
Sequence CWU 1
1
1016210DNAStreptomyces geldanus 1tccgacgagc cggtggcgat cgtggcgatg
ggctgccacc tgccgggcga ggtcgcgacg 60cccgaggacc tgtggcggtt ggtggccgac
gggcgggacg cgatcgccgg gttcccggag 120gaccggggct gggacctggc
cgggctcttc gactccgacc cggacgccgt gggcaagtcc 180tatgtgcgcg
agggcggttt cctcaccgac gcgggcggat tcgacgccgc attcttcggc
240atctcgcccc gtgaggcgct ggcgatggac ccgcagcagc ggttgctgct
ggagaccgcg 300tgggagacct tcgagaatgc cggaatcgac ccgggttcgc
tgcacggcac cgacgtcggt 360gtgttcagcg gagtgatgta ccacgattac
ggggccgacg ccgggacggc ggcggagggc 420ctggaggggc atctcggcgt
gggcagcgcg gggagcgtcg tctccgggcg ggtggcctac 480gcgctgggcc
tgaccgggcc cgcggtgacc gtggacaccg cctgctcgtc ctccctggta
540gcgctgcacc tggcggttca ggcggtgcgc acgggcgaat gctcgctggc
gctcgccggg 600ggtgtcgcgg tgatgagcag gccgacgtcg ttcatcgagt
tctcccgcca gcgtggcctc 660gcccccgacg gccgctgcaa gtccttcgcg
gagggcgccg acggcaccaa ctggtccgag 720ggtgtcgggt tggtgttgct
ggagcggctg tccgatgccc gccgcaatgg gcatgaggtg 780ctcgccgtcg
tccgtggcac cgccgtgaac caggacggcg ccagcaacgg cctgaccgcg
840cccaacggcc cgtcccagga acgggtgatc cggcaggcgc tggcgaacgc
cgggctgacg 900gtggccgatg tggacgcggt cgaggcccac ggcacgggca
cgagtctcgg cgacccgatc 960gaggcccagg cactcctggc cacctacggg
caggagcggc cggaggatca gccgctgtgg 1020ctggggtcgt tgaagtcgaa
catcgggcat gcgcaggcgg cggcgggcgc ggccggtgtc 1080atcaagatgg
tccaggccat gcggcacggc gtactgccca aaaccctcca cgccgacgag
1140cccaccagca aggtcgactg gacgtcaggt gcggtgtcgc tactgtccga
ggcccggccc 1200tggccggaga cgggacaccc ccgccgcgcc ggaatctcct
ccttcggcgt cagcgggacg 1260aacgcacacg tggtcctgga acaggcaccc
ctggaagcgg ctgcacccga aacacaggcg 1320agcgacgcgg gcgctcctgg
gctcgtggcc acgggcggcg tagtgccgtg ggtgctgtcc 1380gccaagactc
ctgcggcgct gcgcgctcag gcagagcgtc tggtcagcca tctggagtcc
1440gggagcgacg ccaacccggt cgatgtgggc tggtcgctgg ccaccacccg
ggcggcgttg 1500gagcaccgcg cggtcatcct ggcgacggat gccgaaggag
gcatggcgac ggcgcgggct 1560ctggcggagg ggcggcctga cccgctcctg
gtcaccggac agaccggaac agacggcaaa 1620accgtgttca tcttccccgg
ccaaggcgcc caatgggtgg gcatgggagc ccaactcctc 1680aacacctcac
ccgtcttcgc cgcccgcctg cgtgagtgcg ccgatgctct agcgccgtat
1740accgactggt cgctcatcga cgtcatcacc ggcacgcccg acgctccctc
gcttgagcgt 1800gtcgacgtcg tacagcccgc caccttcgcc gtcgtcgtct
ccctcgccgc actctggcaa 1860tccgtgggca tccaccccga cgccgtcatc
ggccactccc aaggcgaaat cgccgccgcc 1920tgcgtcgccg gacacctcac
cctcaccaac gccgccaaaa tcgtcaccct ccgcagccag 1980accatcgccc
accacctcgc cggacacggc ggcatgatgt ccctcgccac ccccgccgac
2040accatcgacc tcaccaactg gcacggcaaa ctctggatcg ccgcacacaa
cagccccaac 2100gccaccgtca tcgcaggcga caccgacgcc ctgcaccaac
tccacaccca ctacaccgac 2160cagggcacca gagcccgcat catccccgtc
gactacgcct cccacaccgg acacgtcgac 2220accatcaaaa accagctaca
agacgtactc gacggcatca ccctcgaacc cggcaccatc 2280ccctggctct
ccaccgtcac cggacagtgg atcgaaccca acaccgtcgg cgacagctac
2340tggtaccgca acctccgcca aaccgtgcaa ttcgagcaca ccatccacac
cctcgccgac 2400cagggctacc gcacctacat cgaaatcagc ccccaccccg
tcctcaccac cgccatccaa 2460gaaaccctcg aagccaacga cacccccaac
accaccatcg tcaccggcac cctccgccgc 2520gacgacgaca cccccacccg
cctcctcacc aacctcgccc acctcaccac caacggaaca 2580ccagtcaact
ggcccaccct cttcacaggc acccaaccca cccgcatccc cctccccacc
2640taccccttcc aacaccacca ctactggctc ccccgcaaca ccagcgcagg
cgatgtgagt 2700gccgtgggcc tccagggcac gggccacccg ctggccgggg
ccgtggtgag cgtgcccgac 2760accgggggtg tgctgctcac cgggcagttg
tcggtggcca cccacccgtg gctggccgac 2820cacgccgtct ccggaacggt
gctgctgccg ggcaccgcga tggccgaact cgccatccgc 2880gccggagacg
agaccgacac ccccaccctg gaagagctgg tcatcggcca gccgatgaca
2940ctgcccgaag acggtgcact acatgtccag gtactggtcg gcggcgtgga
ggacgggcgc 3000cgaggggtgc ggatctactc tcgccccgac gcggcccagg
aacaggaatg gctggagcac 3060gcctcgggca cactcgccac gcagccggac
ggttcggccg agggcggcat ggagaacggc 3120atgcccgagt ggccgccgcc
cggtgtcgag ccgatcgctc tggatgactt ctacgacgac 3180ctcgcccagg
ccgggtatga gtacgggccc gccttccgcg ggctgaaggc ggtctggaag
3240cgcgatggcg aggtgttcgc ggaggccgcg ctgccggagg agcagacgga
cgtcgccggc 3300cggttcggta tccatccggc gctgctggac gccgcgttgc
acgcgagcaa cttctgtgtg 3360cccccggccc cgggccaaac gctcctcccc
ttcgtgtgga acggcgtacg gctgctggcg 3420gcgggagcca cggccgtccg
tgtgcgcgcc cgcgccaccg gcacggactc gttcacgatc 3480agcctgttcg
acagcaccgg ctcccccgtc gcctcggtgg actccctggt gctccgggcg
3540atcagtcccg agcagctcgc tgccgcctcc ggcggtgccg gtcggtccgc
tgatgcgctg 3600ttcacgctgg actggaccga gcaccccacc gccctgggga
ccgaggtttc ctgggccacc 3660ctcggcgatg cccacaccga cgtggacgcc
cacgtggacg cgctcatcgc gggagaggac 3720cggcccgggg ccgtggtcgc
cgacaccgcg gcctgggccg ccggggacac cggcctgccc 3780gcgcgggccc
gggatctggc cgcccgcgcg ctggacctgg tgcagcggtg ggtcggccga
3840cccgaactcg ccgacgtccg gctcgtgttg ctcactcgtg gggcggtgtc
cgtgcacgac 3900accgccgagg tcaccgaccc ggccgccgcc gcgatctggg
gcctggtccg ctccgcccag 3960tccgaacacc cgggccggat cgccctggtg
gacaccgacg acgtgtcgcg ggaggcgctg 4020cccgaggcgg tggcggccgg
cgagccgcaa gtggcgctgc gccgtgggct gctgtgggtg 4080cctcgtctgg
tgcggtcgcc gcagggtctc gccgtacccg cgcacgagca ctggtacctc
4140gacgtctcgg agaagggcag cctggagaac ctggtgctgc ggccggatcc
ggaggccacc 4200gcgccgctgg ccaccggtca ggtccggatc gaggtccgcg
ccgccggtca gaacttccgg 4260gacgtactcg tcgcgctcgg cggcgtggcg
ggtcaggagg gtctgggcgg cgagggtgcc 4320ggggtggtga ccgaggtcgg
gcccggggtc gagggcctgg cggtgggcga ccgggtgatg 4380ggcctgttcc
cgcgctcgtt cggcccgctg gccatcgcgg acgcgcgcac ggtcgcgccg
4440atccccgagg gctggtcgta cgccacggcc gccggggtgc cggtggccta
tctgacggca 4500ctgtacgggc tgcgggacct gggcaccgta cagccgggtg
agacggtgct ggtgcacgcc 4560gccgcgggcg gtgtgggcat ggccgccgtc
cagttggcgc ggcacttcgg cgccaccgtg 4620tacgccaccg cccacccgtc
gaagcaccat gtgctgaccg cgctgggggt gccggagggg 4680catctggcgt
ccagccgcga cctcggtttc gcctcggcgt ttcccgcgct ggatgtggtg
4740ctgaactccc tcaccggcga gtatgtggac gcctcgctgg ggctgctcgg
cacgggtggc 4800cgtttcgtgg agatgggcaa gaacgacatc cgcgatcccg
cctcggtcgc cgcagcacat 4860cccggtgtgg gctatcaggc gttcgacctg
ggaggtgacg cgggccctga ccggatccgg 4920gagctgctcg cggagctggt
ggaactgttc gaggcgggcc ggatcgagcc gcttccgata 4980cggcactggg
acgtcaccca ggcgccgacg gccttccggt ggatgagcca ggggcggcac
5040accggcaaga tcgtgctcac cctcccccga gccctggacc cggacggcac
cgtcctgatc 5100accggtggca ccggaaccct cggcgccacc atcgcccgcc
acgtcgtcac ccaccacggc 5160gcgcgccagt tgctcctcat cagccgtcag
ggtcccgacg cccccggcgc caccgatctc 5220accaccgaac tcaccgaact
cggcgccacc gtccgcatca ccgcctgcga caccgccgac 5280cgcgaccaac
tcgccgcgct cctcgccgac atccccgccg cccaccccct caccgccgtc
5340atccacaccg ccggcgccct ggacgacggt gtcctgaccg cgctcacccc
ggaccgcctc 5400gacaccgtct tccgccccaa ggtcgacgcc gtcacccacc
tccacgacct cacccgcgac 5460caggacctgg ccgcgttcgt catctactcg
tccgccgccg gaacgctcgg caacgcgggg 5520caggccaact acgccgccgc
caatgccttc ctcgacgcct tcgcccagtg gcggcacgcc 5580cgccaccggc
ccgccacttc gctggcgtgg gggctgtgga gcgacaccag cacgctcacc
5640tcgacgatgg acgccaccga cgtacgccgc acacggcggg cgggggtgct
gggcatggac 5700aacgccgagg cgctgcgggt gttcgacacc gggttgcggt
ccgggcggcc cgcgctggtg 5760gccgcgaaga tcgacctcac cgccctgcgc
gcgccggacg ccgagttgtc gccgctgctg 5820cgcggactgg cccgtccggc
gcgccgcacc gcgcgcaccg cggccccggc ggccggtggt 5880ctgtcggggc
agctggccgg gctgtccccc gccgggcagc gggagttcct gctcaacctg
5940gtgcgggcgg aggccgcggt ggtcctcgcc cacgccggtc ctgaggcgat
cgagccgacc 6000gtggcgttca aggagatggg tttcgactcg ctgacggcgg
tcgaactgcg caaccggctg 6060aatgcggcga ccgggctgcg gctccccgcc
acgttgctct tcgaccaccc gactccggct 6120cttctcaccg agctgttcca
taccgagttg ggcggcggcc cggcacccgc cgcggcggcc 6180ccggtgaccg
tgcgtgccgc cgctgacgag 621026360DNAStreptomyces geldanus 2atggctgcgt
cccgggaaga cctggtcaag gcgctgcgta cctcgctgat ggacgccgag 60cggctgaagc
gggagaacga ccggctgatc gccgagtcca ccgaaccggt ggcgatcgtg
120gcgatggcgt gccggctgcc gggtggggtg accgacccgg agtcgctgtg
ggagctggtg 180gacgaggggc gggacgcgat cgggccgttc cccacggatc
gcggctggga cctggagacc 240ctgttcgact ccgatccgga cgccgtgggc
aagtcctacg tacgcgaggc ggggttcctg 300gagggggcgg gcggattcga
cgccgccttc ttcggcatct cgccgcgcga ggccctgtcg 360ctggacccgc
agcagcggct gctgctggag accgcgtggg agaccttcga gcgggcgggg
420atggatccgc ggtcggtgga gggccgggac atcgcggtgt tcgccggggg
cagcggccag 480gggtacggcg gcggtccggg tgaggcgccc aagggcctgg
agggctatct gggggtcggc 540gcttccggca gtgtcatctc cgggcgcgtg
tcgtacacgc tcgggctgac cggtcccgcc 600gtgaccgtgg acaccgcctg
ctcgtcctcg ctggtggccg cccatctcgc cgtgcaggcg 660ctgcggtccg
gcgaatgttc catggcgctg gccggtggtg tcgccgtgat gggccagccc
720accgccttcg tcgagttctc ccggcagcgt ggcctggcgc ccgacgggcg
ctgcaagtcc 780ttcggcgcgg gcgccgacgg caccacctgg tccgaaggtg
tcgggctcgt tctgctggag 840cggctgtcgg acgcccgccg caacggccac
gaagtgctgg ccgtgatccg gggcaccgcg 900gtcaaccagg acggcgcctc
caacggactc accgcgccca acggcccctc ccaggagcgg 960gtgatccgcc
aggccctgtc caacgccggg ctgacggtgg ccgacgtgga cgccgtcgag
1020gcccacggca ccggcaccgc cctcggcgac cccatcgaag cccaggccgt
tctcgccacc 1080tacggccaaa gccgcccgga gggccggccg ctgtggctcg
gctccctcaa gtccaacatc 1140ggccacgcgc aggccgcagc gggcatcgcc
agtgtcatca agaccgtcat ggccttacgc 1200cacggccggt tgccgaagac
cctccacgcc gaacagccca cctcccaggt gaactggacg 1260tcgggcgcgg
tgtccctgct cgccgaggcg cgggcgtggc cggagaccgg acacgcccgc
1320cgcgccggga tctcctcctt cggcgtcagc gggacgaacg cacacgtcat
cctggaacag 1380gcccctgagg aagccgaggc gaccggggag aacaccgccg
atcaggaacc gcccgtacgc 1440tcggcggagt ccgccgaccc cggcccggtc
gccaccggcc acgtggtgcc gtggctgctc 1500tcgggccata cgcaggaggc
gctgcgtgcc caggccgccc ggctgctgac ccaggtgcgc 1560gagacgccct
ccgacagtcc gcgggacgtg ggctggtcac tggccaccac ccggacccgg
1620ctggaccacc gcgcggtcgt actgtgcgcc gatgccgagc aggccgtcgc
ggggctggag 1680gcggtggcct cgggcacgtc cgcccggtcg gcggtcaccg
ggtccgtggc ctccggaaag 1740gtggcggtgc tgttcaccgg gcagggcagc
cagcgggccg gaatgggccg cgaactgcac 1800ggcgcccacc cggtgttcgc
gcgggccttc gacgccgtgt gcgcccagtt cggcgacctg 1860cgcgacgggg
acgacaaggt ctcgctggcc gaggtgatct tcgccgagga ggggtcggcg
1920acggcagcgc tgctggaccg gaccgagttc acccagcccg cgctgttcgc
gctggaggtg 1980gcgctgttcc ggctcgtgga gtcgtgggga gtgcgccccg
cgtatgtgct gggccactcg 2040atcggcgaag tggcggcggc ccatgtggcc
ggggtcctgt ccctgccgga cgcctgcaca 2100ttggtgcggg cgcgcgggcg
gctgatgcag caactcaccg cgaccggggc gatggtcgcg 2160gtggaggcgg
ccgaggacga ggtggcgccg ctgctcgcgg ggaaggagca caaggtctcc
2220atcgccgcgg tcaacggccc ggcctccgtg gtcgtctccg gtgacgagga
cgtggtcacg 2280gcggtggcgg agacgctggc gcggcagggc cgcaagacca
agcggctcgt ggtctcgcac 2340gccttccact ccccccacat ggacgggatg
ctggacgcgt tccgcgaggt ggcgtcgcgg 2400ctggcctacg cgccaccccg
gatacccgtg gtgtcgaacc tcaccggcgc ggtcgccgat 2460cccgaggagc
tgtgctcccc cgagtactgg gtacggcatg cacgtggcgc ggtgcggttc
2520ctcgacggtg tccgcacact ggccgacgag ggcgtgcgca cccatctgga
actcggcccg 2580gatggggtgc tgaccgcgat ggggcaggac tgtctgcccg
aggcggacgc ggcgttcgtg 2640ccgtccctgc gtccgggcgt ccaggagccg
cacgcggtgc tggccgggct cgccggcctg 2700tacgtacggg gtgtgcgggt
ggactgggac gcgatgttcg ccgggtccgg cgcccggccc 2760gtcgcccttc
ccacgtacgc cttccagcac gagcactact ggctggagcg ggccgccggc
2820tccggcgacg tgggcgcggt ggggctcggc gaggcgggcc atccgctgct
gggcgcggtg 2880gtgcagctcc cggagacggg cggggtgcag ctcagcgggc
ggctgtcggt acgggcccag 2940ccctggctgg gcgaacacgt catctccggg
gcggtgctgg tgcccggcac cgccatggtg 3000gaactggccg tccgcgccgg
ggacgagacc ggcaccccgg tgctggagga gctggtgatc 3060gggcagccga
tggtgctgcc cggcgacacc gccctcagtg tccaggtcgt cgtgggcgcg
3120gacgagggcg ggcggcgtac ggtgcggatc tactcccgta ccgacggggg
caccgactgg 3180accgagcacg ccaccggcac gctcgcggcg cagggcccgg
caccgctgga cggggccgcg 3240ggcggggccg ccgtcgagtg gccgcccgcg
gaagccgagc cgatccccgt ggaggacttc 3300taccgctcgc tcgtcgacgc
cggatacgcg tacggaccgg cgttccgcgg gctcgtcgcc 3360gcgtggcgcc
gggacggtga gatcttcggc gatgtggcgc tgccggaggc gtccgtcgcg
3420gaggccgagc ggttcggcat ccacccggcg ctgctggacg ccgcactgca
cgcgggcagc 3480ttctgtctgc cctccgaccc ggcgcgacag gtgaccctgc
tgccgttcgc ctggaacacc 3540gtgcgtctgc acgcgggcgg cgcgtccgcg
gtccgggtgc atgtccgccc ggtcggcgac 3600gacgccttct cggtacgcct
gaccgacggc tcgggccaga cggtggcctc ggtggactcg 3660ctcaccttgc
gggcggtgga cccggcccag ctcaagatcg gcacggccga cgacgcgctg
3720tggacggtcc gctggagcga gacctcgctg ccggacggcg cggtctcctg
ggccccgctc 3780ggcgagtcgg ccaccggggc aaccgggggc tacggcgcca
caggggacgg cggaggccca 3840gggggcgcgc ttcccgacgt cctcgtggcc
gatacgcgcg cctgggccga agacctcacc 3900ggaccgccga ccgcgcgggc
ccgggagctc accggccgcc tgctggagga gatccagcgg 3960tgggtcgccg
acgacgccat ggccgggacg cggctcgccg tggtcacccg cggcgcggtc
4020gcggtccacg acgacaccga ggtcaccgac ccggccgcca ccgcgctctg
gggcctggtc 4080cgctcggccc aggccgaaca cccggggcgg gtggccctgg
tggatgccga cggagcgtgc 4140gaggaactgc ccgccggggt gtggtccggg
gacgagcccc aactggcggt gcgcggtggc 4200gccgtgtggg tgccacgcct
cacccgggtc gagcccggcc tgcgcgtgcc cgcgcaggcg 4260tcgtggcatc
tggactcggc cgagtacggc accctggaca atctggcgct gctgcccgac
4320gaggccgagc ccgcaccgcc ggcggccggt caggtgcgga tcgaggtccg
cgccgccggg 4380ctcaacttcc gggatgtcct ggtggctctc ggcatgtatc
cgggccggtc ggtgatcggc 4440acggagggcg ccggtgtggt gaccgaagtc
ggtccgggcg tcacgggcct ggccgtgggc 4500gaccgggtga tgggcctgtt
ctccggctcg ttcggaccgc tggccaccgc cgacgcgcgc 4560acggtgatcc
ggatgccgga gggctggtcg ttcggcacgg cggccggggt gccggtggcc
4620tatctgacgg cgctgtacgc gttgcaggac ctcgggaggg tccagccggg
cgagacggtc 4680ctggtgcacg ccgccgcggg cggtgtgggc atggccgccg
tccagctcgc acagcacttc 4740ggcgccaccg tcctgggcac cgcccacccc
tccaagcacc acgcactcca ccggctgggc 4800gttcccgccg aacggctcgc
ctccagccgc gacctcgcct acgccgacac cttccccacc 4860gccgacgtcg
tcctcaactc cctcaccggc gagcacatcg acgcctccct cggacttctc
4920aaccccggcg gccggttcct ggagatgggg aagaccgacc tgcgggagcc
cggcgaggtc 4980ggggcgcggc atccggaggt cacctaccgg gcgttcgatc
tcggtgggga ggcccccgcg 5040gagcgggtgc gggagttgct gcaccagttg
gtggagctgt tcgaggcggg ccggatcgag 5100ccgctgccgg tacggcagtg
ggacatcacc cgcgcccccg aggcgttccg ctggatgagt 5160caggggcggc
ataccggcaa gatcgtgctc accctgccac gcgccctgga cccggacggc
5220accgtcctgg tcaccggtgg cacgggcacc ctcggcgcca cgatcgcccg
ccaccttctc 5280acccagcacg gcgcacgcca tctgctgctg gtcagccgcc
ggggaccgga cgcacctggc 5340gccacagacc tgaccaccga actcaccgaa
ctcggcgcca ccgtccgcat caccgcctgc 5400gacaccgccg accgcgacca
actcgccgcg ctcctcgccg acatccccgc cgaccacccc 5460ctcaccgccg
tggtccacac ggccgggacc ctcgacgacg gtgtcctgac cgcgctcacc
5520ccggaccgcc tcgacaccgt cttccgcccc aaggtcgacg ccgtcaccca
tctccacgac 5580ctcacccgcg accacgacct ggcggcgttc gtggtgtact
cgtccgccgc cggagtcctc 5640ggcgggcccg gccagggcaa ctactccgcc
gccaacgcct atctggacgg actcgcacag 5700tggcggcgtg cgcacgggct
ccccgccacc tcgctggcgt ggggcatgtg ggcgcagacc 5760agtggcatga
cggccgggct cggctccggc gatctgcacc gggtgcggcg tggcggcatc
5820gtcgggctgt ccacggcgga ggccctggac ctgttcgacc ggtcggtggc
gtccgggctg 5880tccctgctgg tgccgttgcg gttggacatc gccgccctcg
gtgcggaggc cgcggaaccg 5940ccgccgctgc tgcggggtct ggtccggccg
gcccggcgta cggcccggcc ggtgccgaag 6000gccggtgagg gcggcctcgc
cgaacggctg gccgggctgt cggcggccga acaggagcgt 6060ctgctcatcg
agttgatccg cgaacaggcc gcttcggtgc tcgggttccc cacggtcgac
6120ccgatcgggc cggagcaggc gttccgcgac atggggttcg actcgctgac
cgcggtggag 6180ctgcgcaacc gcctcaacac ggccaccggg ctacggctcc
ccgcaacgct ggtcttcgac 6240cacccgagcc ccttggccac cgccgagttc
ctgcgggatc aactgggcgg gcgcgcggtc 6300gaggcggcgc cccgcccggc
ccggcgtgac cggtcggctc cggacggggc cgaggatccg 636034PRTArtificial
SequenceSynthetically constructed specificity peptide sequence of
module 1 AT domain 3Tyr Ala Ser His1412DNAArtificial
SequenceSynthetically constructed specificity nucleotide sequence
of module 1 AT domain 4tacgcctccc ac 1254PRTArtificial
SequenceSynthetically constructed specificity peptide sequence of
module 1 AT domain 5His Ala Phe His1612DNAArtificial
SequenceSynthetically constructed specificity nucleotide sequence
of module 1 AT domain 6cacgccttcc ac 12710PRTArtificial
SequenceSynthetically constructed signature peptide sequence of
module 6 DH domain 7His Val Ile Ser Gly Ala Val Leu Val Pro1 5
10830DNAArtificial SequenceSynthetically constructed signature
nucleotide sequence of module d DH domain 8cacgtcatct ccggggcggt
gctggtgccc 30910PRTArtificial SequenceSynthetically constructed
signature peptide sequence of module 6 DH domain 9His Ala Val Ser
Gly Thr Val Leu Leu Pro1 5 101030DNAArtificial
SequenceSynthetically constructed signature nucleotide sequence of
module 6 DH domain 10cacgccgtct ccggaacggt gctgctgccg 30
* * * * *