Note: Descriptions are shown in the official language in which they were submitted.
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Title
P~ARM~CEUTICAL COMPOUNDS
F;eld Of The Invention
This invention relates to the fields of
pharmaceutical and organic chemistry and provides novel
cryptophycin compounds useful as anti-microtubule agents.
.R~around Of The Tnvention
Neoplastic diseases, characterized by the
proliferation of cells not subject to the normal control of
cell growth, are a major cause of death in humans and other
m~mm~ls. Clinical experience in cancer chemotherapy has
demonstrated that new and more effective drugs are desirable
to treat these diseases. Such clinical experience has also
demonstrated that drugs which disrupt the microtubule system
of the cytoskeleton can be effective in inhibiting the
proliferation of neoplastic cells.
The microtubule system of eucaryotic cells is a
major component of the cytoskeleton and is a dynamic assembly
and disassembly. Thus heterodimers of tubulin are
polymerized and form microtubule. Microtubules play a key
role in the regulation of cell architecture, metabolism, and
division. The dynamic state of microtubules is critical to
their normal function. With respect to cell division,
tubulin is polymerized into microtubles that form the mitotic
spindle.
SUBSTITUTE SHEET (RULE 26)
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-2-
The microtubules are then depolymerized when the mitotic
spindle~s use has been fulfilled. Accordingly, agents which
disrupt the polymerization or depolymerization of
microtubules, and thereby inhibit mitosis, comprise some of
the most effective cancer chemotherapeutic agents in
clinical use.
Additionally, the compounds claimed herein possess
fungicidal properties. Further, such agents having the
ability to disrupt the microtubule system can be useful for
research purposes.
Certain cryptophycin compounds are known in the
literature; however, cryptophycin compounds having even
greater solubility, robust potency are desired for most
pharmaceutical uses and a broader library of cryptophycin
compounds could provide additional treatment options.
Applicants have now discovered novel compounds providing such
desired solubility as well compounds having the ability to
disrupt the microtubule system. Such compounds can be
prepared using total synthetic methods and are therefore well
suited for development as pharmaceutically useful agents.
Sununarv Of ~he Invention
The presently claimed invention provides novel
cryptophycin compounds of Formula I
Rl R3 R5
Ar ~ ~R4 1 0
o~ HN~ ~R6
R9--y~--N ~
wherein
Ar is phenyl or any simple unsubstituted or substituted
aromatic or heteroaromatic group, C1-C12 alkyl, C1-C12 alkyne;
SUBSTITUTE SHEET (RULE 26)
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R1 is halogen, SH, amino, monoalkylamino, dialkylamino,
trialkylammonium, alkylthio, dialkylsulfonium, sulfate, or
phosphate;
R2 is OH or SH; or
R1 and R2 may be taken together to form an epoxide ring, an
aziridine ring, an episulfide ring, a sulfate ring, a
cyclopropyl ring, or monoalkylphosphate ring; or
R1 and R2 may be taken together to form a second bond between
C1g and C1g;
R3 iS a lower alkyl group;
R4 iS H;
R5 is H;
R4 and R5 may be taken together to form a second bond between
C13 and C14;
R6 is a substituent selected from the group consisting of B-
ring heteroaromatic, substituted heteroaromatic, B-ring
(C1-C6)alkyl, (C3-Cg)cycloalkyl, substituted C3-Cg cycloalkyl,
substituted (C1-C6)alkyl, a group of the formula III~:
D16
Z~I~R15
Rl7 III' and a group of the formula III'':
R16
\~\
Z~
R15
R17 III'';
R7 iS selected from the group consisting of MR51R52,
R53NR51R52, oR53, H and a lower alkyl group; R51 and R52 are
independently selected from the group consisting of C1-C3
alkyl; R53 iS Cl-C3 alkyl;
R8 is H or a lower alkyl group;
R7 and R8 can optionally form a cyclopropyl ring;
S~Jb~ 111 ~JTE SHEET (RULE 26)
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R9 is selected from the group consisting of H, a lower alkyl
group, unsaturated lower alkyl, and lower alkyl-C3-Cs
cycloalkyl;
R10 iS H or a lower alkyl group;
R9 and R10 together optionally form a cyclopropyl ring;
R11 is selected from the group consisting of H, OH, simple
alkyl, phenyl, substituted phenyl, benzyl, and substituted
benzyl;
R14 iS H or a lower alkyl group;
R15, R16, and Rl7 are each independently selected from the
group consisting of hydrogen, (C1-C6)alkyl, ORl8, halo,
NR13 Rl9 , N02, OP04H2, OR19phenyl, SCH2phenyl, CONH2, C02H,
PO3H2, and So2R23, and ZZ;
R13 is selected from the group consisting of hydrogen, aryl,
and C1-C6 alkyl;
R13' is selected from the group consisting of hydrogen and
(C1-C6)alkyl;
R19 is C1-C6 alkyl;
R19' is selected ~rom the group consisting o~ hydrogen and
(C1-C6)alkyl;
R23 is selected from the group consisting of hydrogen and (Cl-
C3 ) alkyl;
R30 is hydrogen or C1-C6 alkyl;
n is 0, 1, or 2;
p is 0, 1, or 2;
m is 0, 1, or 2;
X is selected from the group consisting of 0, NH and
alkylamino;
Y is selected from the group consisting of 0, NH, and
alkylamino;
Z is selected from the group consisting of -(CH2) n~
- ( CH2 ) p-O- ( CH2 ) m~ and ( C3 -Cs ) cycloalkyl;
ZZ is selected from the group consisting of an aromatic group
and a substituted aromatic group; or
a pharmaceutically acceptable salt or solvate thereof;
provided that when R6 is a group of Formula III' and n is 1,
then at least one of the group consisting of R15, R16 and R17
Sl._S 1 1 1 UTE SHEET (RULE 26)
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must be a non-hydrogen group and if only one of Rl5, R16 and
Rl7 is OH or oR29 and one of the group consisting of R15, R16
and Rl7 is halo then the remaining member of the group
consisting of R15, R16 and R17 must not be hydrogen or halo;
R29 is (Cl-Cs)alkyl;
further provided that the compound is not a cryptophycin
selected from the group consisting of cryptophycins:
O H~ ~ ~ Br
O ~ N ~ O ~ OCH3
B-2
o H~ ~ ~ Cl
H OCH2CH3
SUBSTITUTE SHEET (RULE 26)
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o
N ~ O ~ OCH3
C-l
~~
~ ~ H ~ ~ Col H3
C-2
~,1 , ,~
~ ~ ColH3
C-3
SU~;~ JTE SHEET (RULE 26)
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'JD ~ o
O ~ N ~ ~ ~ Col H3
Et Et
C-6
'~D ~
O /~ N~ O~COCH 3
CRYPTO PHYC IN - 5 2
~ O
~o~Nio \~OCH3
CRYPTOPHYCIN-210
SUBSTITUTE SHEET(RULE26)
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N ~ ~ OCH3
CRYPTOPHYCIN-190
~ ~ ~ ~ OCH3
CRYPTOPHYCIM-189
~
o H~ ~ ~ Cl
O ~ N ~ O ~ OCH3
H
CRYPTOPHYCIN-115
SUBSTITUTE SHEET (RULE 26)
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~= O
~ ~ "~C 1
O ~ M ~ O ~ OCH3
CRYPTOPHYCIN-110
~~
O H~ ~ ~ Cl
HM OCH3
Crypto~hycin-215
~ 0
~ ~ M ~ ~ ColH3
Crypto~hycin-214
Sl)a;~ JTE SHEET (RULE 26)
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--10--
O ~ N ~ ~ OCX3
Crypto~hycin-213
r~
o HN ~ ~ Cl
O ~ N ~ O ~ OCH3
Crypto~hycin-211 and
~
q,P ~ ~,c 1
O ~ N ~ ~ OCH3
/\ ~
D-2
The present invention provides pharmaceutical
formulations, a method for disrupting a microtubulin system
using an effective amount of a compound of Formula I, a
method for inhibiting the proliferation of m~mm~l ian cells
comprising administering an effective amount of a compound of
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--11--
Formula I, and a method for treating neoplasia in a mammal
comprising administering an effective amount of a compound of
Formula I.
~etailed DescriDtion of the Invention
As used herein, the term ~Isimple alkyl" shall refer
to Cl-C7 alkyl wherein the alkyl may be saturated,
unsaturated, branched, or straight chain. Examples include,
but are in no way limited to, methyl, ethyl, n-propyl, iso-
propyl, n-butyl, propenyl, sec-butyl, n-pentyl, isobutyl,
tert-butyl, sec-butyl, methylated butyl groups, pentyl, tert
pentyl, sec-pentyl, methylated pentyl groups and the like.
As used herein, the term "B-ring C1-C6 alkyl~
refers to saturated, unsaturated, branched and straight chain
alkyl wherein the B-ring C1-C6alkyl group may include up to
three (3) non-carbon substituents. Such non-carbon
substituents are most preferredly selected from the group
consisting of OH, SCH2phenyl, NH2, CO, CONH2, C02H, PO3H2,
SO2R21 wherein R21 is selected from hydrogen and C1-C3 alkyl;
As used herein, the term ~substituted phenyl~ shall
refer to a phenyl group with from one to three non-
hydrocarbon substituents which may be independently selected
from the group consisting of simple alkyl, Cl, Br, F, and I.
As used herein, the term '~substituted benzylll shall
refer to a benzyl group with from one to three non-
hydrocarbon substitutents which may be independently selected
from the group consisting of simple alkyl, Cl, Br, E, and I
wherein such substituents may be attached at any available
carbon atom.
As used herein "B-ring heteroaromatic group~ refers
to aromatic rings which contain one or more non-carbon
substituent selected from the group consisting of oxygen,
nitrogen, and sulfur. Especially preferred B-ring
heterocyclic groups are selected from, but not limited to,
the group consisting of
SUBSTITUTE SHEET (RULE 26)
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~- oR20 E~ ~N
0>
wherein R20 is selected ~rom hydrogen and Cl-C6 alkyl.
It is especially preferred that "B-ring
heteroaromatic group" re~ers to a substituent selected from
the group consisting o~:
~= OR~ />
~ ~oR20
As used herein ~Icycloalkylll re~ers to a saturated
Cl-C8 cycloalkyl group wherein such group may include from
zero to three substituents selected from the group consisting
SUBSTITUTE SHEET(RULE 26)
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of Cl-C3 alkyl, halo, and oR22 wherein R22 is selected from
hydrogen and Cl-C3 alkyl. Such substituents may be attached
at any available carbon atom. It is especially preferred
that cycloalkyl refers to substituted or unsubstituted
cyclohexyl.
As used herein ~Lower alkoxyl group" means any
alkyl group of one to five carbon atoms bonded to an oxygen
atom. As used herein ~lower alkyl group" means an alkyl
group of one to five carbons and includes linear and non-
linear hydrocarbon chains, including for example, but not
limited to, methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, tert-butyl, sec-butyl, methylated butyl groups,
pentyl, tert pentyl, sec-pentyl, and methylated pentyl
groups. As used herein "allylically substituted alkene~
means any alkene having from one to seven carbon atoms which
contains an alkyl substitution on it. As used herein the
term "unsaturated lower alkyl'~ means a lower alkyl group as
defined supra . wherein from one to two double bonds are
present in the unsaturated lower alkyl substituent. A
pre~erred unsaturated lower alkyl is -CH2-CH=CH2. The term
~lower alkyl-C3-Cs cycloalkyl~ refers to C-C alkyl substituted
with a C3-Cscycloalkyl group. A preferred lower alkyl-c3-cs
cycloalkyl group is -CH2-cyclopropyl; wherein the group is
attached to the cryptophycin core structure at R9 via the CH2
As used herein ~epoxide ring" means a three-
membered ring whose backbone consists of two carbons and an
oxygen atom. As used herein, "aziridine ring" means a three-
membered ring whose backbone consists of two carbon atoms and
a nitrogen atom. As used herein ~sulfide ring" means a
three-membered ring whose backbone consists of two carbon
atoms and a sulfur atom. As used herein "episulfide ring~
means a three-membered ring whose backbone consists of two
carbon atoms and a sulfur atom. As used herein "sulfate
group~ means a five membered ring consisting of a carbon-
carbon-oxygen-sulfur-oxygen backbone with two additional
oxygen atoms connected to the sulfur atom. As used herein
~'cyclopropyl ring" means a three member ring whose backbone
SIJ~;~ JTE SHEET (RULE 26)
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-14-
consists of three carbon atoms. As used herein,
"monoalkylphosphate ring~' means a five membered ring
consisting of a carbon-carbon-oxygen-phosphorous-oxygen
backbone with two additional oxygen atoms, one of which bears
a lower alkyl group, connected to the phosphorous atom.
As used herein, "simple unsubstituted aromatic
group" refers to common aromatic rings having 4n+2 electrons
in a monocyclic conjugated system, for example, but not
limited to: furyl, pyrrolyl, thienyl, pyridyl and the like,
or a bicyclic conjugated system, for example but not limited
to indolyl or naphthyl.
As used herein "simple substituted aromatic group'~
refers to a phenyl group substituted with a single group
selected from the group consisting of halogen and lower alkyl
group.
As used herein, llheteroaromatic group" refers to
aromatic rings which contain one or more non-carbon
substituent selected from the group consisting of oxygen,
nitrogen, and sulfur.
As used herein, ~halogenll or "halo" refers to those
members of the group on the periodic table historically known
as halogens. Methods of halogenation include, but are not
limited to, the addition of hydrogen halides, substitution at
high temperature, photohalogenation, etc., and such methods
are known to the skilled artisan.
As used herein, the term ~mAmmAl~ shall refer to
the M~mmAlia class of higher vertebrates. The term "mAmmAl "
includes, but is not limited to, a human. The term
"treating~ as used herein includes prophylaxis of the named
condition or amelioration or elimination of the condition
once it has been established. The cryptophycin compounds
claimed herein can be useful for veterinary health purposes
as well as for the treatment of a human patient.
Some preferred characteristics of this invention
are set forth in the following tabular form wherein the
features may be independently selected to provide preferred
SUBSTITUTE SHEET (RULE 2~)
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embodiments of this invention. The invention is in no way
limited to the features described below:
A) R8 is ethyl, propyl, isopropyl, butyl, isobutyl
or isopentyl;
B) R7 is ethyl, propyl, isopropyl, butyl, isobutyl,
pentyl, or isopentyl;
C) R7 iS H, R8 is methyl, R3 is methyl, and X and Y
are not both O;
D) R3 iS ethyl, propyl, isopropyl, butyl, isobutyl,
pentyl or isopentyl;
E) R9 iS methyl, ethyl, propyl, butyl, isobutyl,
pentyl, or isopentyl;
E) Rl~ is methyl, ethyl, propyl, butyl, isobutyl,
pentyl, or isopentyl;
G) a cryptophycin compound wherein at least one of
the groups selected from the group consisting of C-
3, C-6, C-7, C-10, C-16, C-17, and C-18 has R
stereochemistry (numbering as set forth in Formula
I supra. );
H) a cryptophycin compound wherein at least one of
the groups selected from the group consisting of C-
3, C-6, C-7, C-10, C-16, C-17, and C-18 has S
stereochemistry (numbering as set forth in Formula
I supra. );
I) Ar is phenyl with a substituent selected from
the group consisting of hydrogen, halogen, and
simple alkyl;
J) a compound wherein Y is O;
K) a compound wherein Y is O, R7, R8, R9, and R10
are each hydrogen; and Rl and R2 form an epoxide;
L) R7, R8 are each hydrogen;
M) R7 and R8 are each selected from hydrogen and
CH3;
N) Y is O;
O) R is selected from the group consisting of
methyl, ethyl, n-propyl, and phenyl;
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p) Rl and R2 form an epoxide ring;
Q) both x and Y are O
R) R4 and R5 form a double bond;
S) n is O; R6 is substituted benzyl wherein one
substituent is a halogen and one is an OR12 group
wherein R12 iS lower alkyl;
T) a compound of Formula I is used for disruption
of a microtubulin system;
U) a compound of Formula I is used as an anti-
neoplastic agent;
V) a compound of Formula I is used for the
treatment of cancer in a m~mm~ l;
W) a compound of Formula I is used as an antifungal
agent;
x) R6 is Formula III' and is para hydroxy
substituted;
Y) R6 is selected from the group consisting of
SUBSTITUTE S!.~ RULE 26)
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-17-
~OH
~~ ~
~NH
--~OH --~ N
SUBSTITUTE SHEET (RULE 26)
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-18-
/--~NI lZ /--
Nt
Z) Z is - (CH2 ) n~ wherein n is 0;
AA) Z is -(CH2) n~ wherein n is 2;
BB) Z is -(CH2) n~ wherein n is 1;
CC ) R6 is Formula III';
DD) R6 is Formula III'';
EE) R6 is C3-C6 cycloalkyl;
FF) R6 is selected ~rom the group consisting o~ B-
ring heteroaromatic, substituted heteroaromatic, s-ring
10 alkyl, cycloalkyl, substituted cycloalkyl, Formula III' and
Formula III'';
GG) at least one o~ Rl5, Rl6, and Rl7 is selected
from the group consisting o~ SCH2phenyl, NH2, CO, CONH2, CO2H,
PO3H2, and S02R2l; wherein R21 is selected ~rom hydrogen and
15 Cl-C3 alkyl;
HH) Ar is phenyl;
II) Ar is phenyl substituted with one or two ~rom
the group consisting o~ OH, OCH3, halo, and methyl; and
JJ) Ar is naphthyl;
KK) R6 has a Z wherein the ~irst carbon o~ the Z
group is with respect to the point o~
attachment to the cryptophycin molecule;
LL) R6 is a heteroaromatic ring;
SUBSTITUTE SHEET (RULE 26)
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--19--
MM) R7 is selected from the group consisting of
N(CH3)2, CH2N(cH3)2i
NN) R7 is CH2OCH3;
Oo) R7 is cyclopropyl;
PP) R9 is CH2cyclopropyl;
QQ) R9 is CH2cH=cH2
To further illustrate, but to no way limit, the
compounds contemplated herein, the following table of
especially preferred compounds is provided:
A compound wherein R3 is CH3; R4 and R5 together form a second
bond; Rl4 is hydrogen; R30 is hydrogen; R7 and R8 are each
methyl; R10 is hydrogen; R9 is -CH2CH(CH3)2; X and Y are each
O; Ar is phenyl; and
~1 R2 R6
\
~,
together form a double bond
together form an epoxide OPO4H~
together form an epoxide
,. \
together form a double bond
SUBSTITUTE SHEET (RULE 26)
.
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-20-
,... ~
C1 OH I ,
'"'"""""~\/¢~
Cl OH
""'"""""~\/\~
together form a double bond
"""""
"""\/~
together form an epoxide
~c
-'"""
"""~\/~
C1 OH OH ,
together ~or a double oond ~ / V ~ 1
SUIBSTITUTE SHEET (RULE 26)
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Cl OH ~ /
together form a double bond
...",.~n~
together form an epoxide
CI
l! I
C 1 OH ~ opo3H2
Additional preferred compounds are those named above except
Il I
that Ar is ~OCH3 instead of phenyl-
Further preferred compounds are those named above except that
Ar is OCH3
The present invention provides a method of
alleviating a pathological condition caused by
hyperproliferating mammalian cells comprising administering
to a subject an e~fective amount of a pharmaceutical or
veterinary composition disclosed herein to inhibit
SUBSTITUTE SHEET (RULE 26)
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proliferation of the cells. In a preferred embodiment of this
invention, the method further comprises administering to the
subject at least one additional therapy directed to
alleviating the pathological condition. In a preferred
embodiment of the present invention, the pathological
condition is characterized by the formation of neoplasms. In
a further preferred embodiment of the present invention, the
neoplasms are selected from the group consisting of m~mm~y,
small-cell lung, non-small-cell lung, colorectal, leukemia,
melanoma, pancreatic adenocarc; nom~, central nervous system
(CNS), ovarian, prostate, sarcoma of soft tissue or bone,
head and neck, gastric which includes pancreatic and
esophageal, stomach, myeloma, bladder, renal, neuroendocrine
which includes thyroid and non-Hodgkin's disease and
Hodgkin's disease neoplasms.
As used herein "neoplastic" refers to a neoplasm,
which is an abnormal growth, such growth occurring because of
a proliferation of cells not sub~ect to the usual limitations
of growth. As used herein, ~lanti-neoplastic agent'~ is any
compound, composition, admixture, co-mixture, or blend which
inhibits, eliminates, retards, or reverses the neoplastic
phenotype of a cell.
Anti-mitotic agents may be classified into three
groups on the basis of their molecular mechanism of action.
The first group consists of agents, including colchicine and
colcemid, which inhibit the formation of microtubules by
sequestering tubulin. The second group consists of agents,
including vinblastine and vincristine, which induce the
formation of paracrystalline aggregates of tubulin.
Vinblastine and vincristine are well known anticancer drugs:
their action of disrupting mitotic spindle microtubules
preferentially inhibits hyperproliferative cells. The third
group consists of agents, including taxol, which promote the
polymerization of tubulin and thus stabilizes microtubules.
The exhibition of drug resistance and multiple-drug
resistance phenotype by many tumor cells and the clinically
proven mode of action of anti-microtubule agents against
SlJ~s ~ JTE SHEET (RULE 26)
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-23-
neoplastic cells necessitates the development of anti-
microtubule agents cytotoxic to non-drug resistant neoplastic
cells as well as cytotoxic to neoplastic cells with a drug
resistant phenotype.
Chemotherapy, surgery, radiation therpy, therapy
with biological response modifiers, and immunotherapy are
currently used in the treatment of cancer. Each mode of
therapy has specific indications which are known to those of
ordinary skill in the art, and one or all may be employed in
an attempt to achieve total destruction of neoplastic cells.
Moreover, combination chemotherapy, chemotherapy utilizing
compounds of Formula I in combination with other neoplastic
agents, is also provided by the subject invention as
combination therapy is generally more effective than the use
of a single anti-neoplastic agent. Thus, a further aspect of ==
the present invention provides compositions cont~-ning a
therapeutically effective amount of at least one compound of
Formula I, including the non-toxic addition salts thereof,
which serve to provide the above recited benefits. Such
compositions can also be provided together with
physiologically tolerable liquid, gel, or solid carriers,
diluents, adjuvants and excipients. Such carriers,
adjuvants, and excipients may be found in the U.S.
Pharmaco~eia, Vol. XXII and ~ational Formularv vol XVII,
U.S. Pharmaco~eia Convention, Inc. Rockville, MD (1989).
Additional modes of treatment are provided in AHFS Drua
Information, 1993 e. by the American Hospital Formulary
Service, pp. 522-660. Each of these references are well
known and readily available to the skilled artisan.
The present invention further provides a
pharmaceutical composition used to treat neoplastic disease
containing at least one compound of Formula I and at least
one additional anti-neoplastic agent. Anti-neoplastic agents
which may be utilized in combination with Formula I compounds
include those provided in the Merck Index 11, pp 16-17,
Merck & Co., Inc. (1989). The ~erck Index is widely
recognized and readily available to the skilled artisan
SUBSTITUTE SHEET (RULE 26)
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In a further embodiment of this invention,
antineoplastic agents may be antimetabolites which may
include but are in no way limited to those selected from the
group consisting of methotrexate, 5-fluorouracil, 6-
mercaptopurine, cytosine, arabinoside, hydroxyurea, and 2-
chlorodeoxyadenosine. In another embodiment of the present
invention, the anti-neoplastic agents contemplated are
alkylating agents which may include but are in no way limited
to those selected from the group consisting of
cyclophosphamide, mephalan, busulfan, paraplatin,
chlorambucil, and nitrogen mustard. In a further embodiment,
the anti-neoplastic agents are plant alkaloids which may
include but are in no way limited to those selected from the
group consisting of vincristine, vinblastine, taxol, and
etoposide. In a further embodiment, the anti-neoplastic
agents contemplated are antibiotics which may include, but
are in no way limited to those selected from the group
consisting o~ doxorubicin, daunorubicin, mitomycin C, and
bleomycin. In a further embodiment, the anti-neoplastic
agents contemplated are hormones which may include, but are
in no way limited to those selected from the group consisting
of calusterone, diomostavolone, propionate, epitiostanol,
mepitiostane, testolactone, tamoxifen, polyestradiol
phosphate, megesterol acetate, flutamide, nilutamide, and
trilotane.
In a further embodiment, the anti-neoplastic agents
contemplated include enzymes which may include, but are in no
way limited to those selected from the group consisting of ~-
Asparginase and aminoacridine derivatives such as, but not
limited to, amsacrine. Additional anti-neoplastic agents
include those provided by Skeel, Roland T., "Antineoplastic
Drugs and Biologic Response Modifier: Classification, Use and
Toxicity of Clinically Useful Agents" Handbook of Cancer
Chemothera~v (3rd ed.), Little Brown ~ Co. (1991).
These compounds and compositions can be
administered to mAmm~ls ~or veterinary use. For example,
domestic ~n i m~1s can be treated in much the same way as a
SUBSTITUTE Sl .__ I (RULE 26)
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-25-
human clinical patient. In general, the dosage required for
therapeutic effect will vary according to the type of use,
mode of administration, as well as the particularized
requirements of the individual hosts. Typically, dosages
will range from about 0.001 to 1000 mg/kg, and more usually
0.01 to 10 mg/kg of the host body weight. Alternatively,
dosages within these ranges can be ~mini stered by constant
infusion over an extended period of time, usually exceeding
24 hours, until the desired therapeutic benefits are
obtained. Indeed, drug dosage, as well as route of
administration, must be selected on the basis of relative
effectiveness, relative toxicity, growth characteristics of
tumor and effect of Formula I compound on cell cycle, drug
pharmacokinetics, age, sex, physical condition of the patient
and prior treatment, which can be determined by the skilled
artisan.
The compound of Formula I, with or without
additional anti-neoplastic agents, may be formulated into
therapeutic compositions as natural or salt forms.
Pharmaceutically acceptable non-toxic salts include base
addition salts which may be derived from inorganic bases such
as for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine,
and the like. Such salts may also be formed as acid addition
salts with any free cationic groups and will generally be
formed with inorganic acids such as for example, hydrochloric
or phosphoric acids or organic acids such as acetic, oxalic,
tartaric, mandelic, and the like. Additional excipients
which further the invention are provided to the skilled
artisan for example in the U.S. Pharmaco~eia.
The suitability of particular carriers for
inclusion in a given therapeutic composition depends on the
pre~erred route of administration. For example, anti-
neoplastic compositions may be formulated for oral
administration. Such compositions are typically prepared as
liquid solution or suspensions or in solid forms. Oral
SU~IllUTE SHEET(RULE 26)
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~ormulation usually include such additives as binders,
fillers, carriers, preservatives, stabilizing agents,
emulsifiers, buffers, mannitol, lactose, starch, magnesium
stearate, sodium saccharin, cellulose, magnesium carbonate,
and the like. These compositions may take the form of
solutions, suspensions, tablets, pills, capsules, sustained
relsease formulations, or powders, and typically contain 1%
to 95% of active ingedient. More preferably, the composition
contains from about 2% to about 70% active ingredient.
Compositions of the present invention may be
prepared as injectables, either as liquid solutions,
suspensions, or emulsions; solid forms suitable for solution
in or suspension in li~uid prior to injection. Such
injectables may be administered subcutaneously,
intravenously, intraperitoneally, intramuscularly,
intrathecally, or intrapleurally. The active ingredient or
ingredients are often mixed with diluents, carriers, or
excipients which are physiologically tolerable and compatible
with the active ingredient(s). Suitable diluents and
excipients are for example, water, saline, dextrose,
glycerol, or the like and combinations thereof. In addition,
if desired, the compositions may contain minor amounts of
auxilary substances such as wetting or emulsifying agents,
stabilizing or pH buffering agents.
The invention further provides methods for using
Formula I compounds to inhibit the proliferation of m~mm~lian
cells by contacting these cells with a Formula I compound in
an amount sufficient to inhibit the proliferation of the
m~mm~lian cell. A preferred embodiment is a method to
inhibit the proliferation of hyperproliferative mammalian
cells. For purposes of this invention ~hyperproliferative
m~mm~lian cells~ are mammalian cells which are not subject to
the characteristic limitations of growth (programmed cell
death for example). A further preferred embodiment is when
the m~mm~lian cell is human. The invention further provides
contacting the m~mm~lian cell with at least one Formula I
SUBSTITUTE SHEET (RULE 26j
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compound and at least one anti-neoplastic agent. The types
of anti-neoplastic agents contemplated are discussed supra.
The invention further provides methods for using a
compound of Formula I to inhibit the proliferation of
hyperproliferative cells with drug-resistant phenotypes,
including those with multiple drug-resistant phenotypes, by
contacting said cell with a compound of Formula I in an
amount sufficient to inhibit the proliferation of a
hyperproliferative mAmmAlian cell. A preferred embodiment is
when the mammalian cell is human. The invention further
provides contacting a Formula I compound and at least one
additional anti-neoplastic agent, discussed supra.
The invention provides a method for alleviating
pathological conditions caused by hyperproliferating
mAmmAlian cells for example, neoplasia, by administering to a
subject an effective amount of a pharmaceutical composition
containing Formula I compound to inhibit the proliferation of
the hyperproliferating cells. As used herein llpathological
condition~ refers to any pathology arising from the
proliferation of mammalian cells that are not subject to the
normal limitations of growth. Such proliferation of cells
may be due to neoplasms as discussed supra.
In a further preferred embodiment the neoplastic
cells are human. The present invention provides methods of
alleviating such pathological conditions utilizing a compound
of Formula I in combination with other therapies, as well as
other anti-neoplastic agents.
The effectiveness of the claimed compounds can be
assessed using standard methods known to the skilled artisan.
Examples of such methods are as follows:
Compounds of this invention have been found to be
useful against pathogenic fungi. For example, the usefulness
for treating Cryptococcus neoformans can be illustrated with
test results against Cryptococcus neoformans employing yeast
nitrogen base detrose agar medium. In carrying out the
assay, a compound of this invention is solubilized in
dimethyl sulfoxide supplemented with Tween 20. Twofold
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dilutions are made with sterile distilled water/10 percent
DMSO to obtain final drug concentrations in the agar dilution
assay plates ranging from 0 .008 ~Lg/ml to 16.0 ~g/ml against
an expanded panel of 84 C~yptococcus neoformans strains. The
m; ni mllm inhibitory concentration against the panel of 84
Cryptococcus neoformans isolates is determined to illustrate
the desired antifungal activity.
The compounds are screened for m;n;mllm inhibitory
concentrations against KB, a human nasopharyngeal carcinoma
cell line, LoVo, a human colorectal adenocarcinoma cell line
using The Corbett assay, see Corbett, T.H. et al. Cvtotoxic
Anticancer Druas: Models and Conce~ts for Drua Discoverv and
Develo~ment, pp 35-87, Kluwer Academic Publishers: Norwell,
1992. see also, Valeriote, et al. l~iscoverv and Develo~ment
of Anticancer Aaents, Kluwer Academic Publishers, Norwell,
1993 is used for the evaluation of compounds.
The most active compounds are further evaluated for
cytotoxicity against four different cell types, for example a
murine leukemia, a murine solid tumor, a human solid tumor,
and a low malignancy fibroblast using the Corbett assay.
The compounds are further evaluated against a broad
spectrum of murine and human tumors implanted in mice,
including drug resistant tumors.
Tumor burden (T/C) (mean tumor burden in treated
~n;m~ls versus mean tumor burden in untreated ;~n;m~ls) are
used as a further assessment. T/C values that are less than
42% are considered to be active by National Cancer Institute
Standards; T/C values less than 10% are considered to have
excellent activity and potential clinical activity by
National Cancer Institute standards.
Materials
Vinblastine, cytochalasin B, tetramethylrhodamine
isothiocvanate (TRITC)-phalloidin, sulforhodamine B (SRB) and
antibodies against ~-tubulin and vimentin are commercially
available from recognized commercial vendors. sasal Medium
Eagle containing Earle's salts (BME) and Fetal Bovine Serum
(FBS) are also commercially available.
SUBSTITUTE SHEET (RULE 26)
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--2g -
Cell Lines
The Jurkat T cell leukemia line and A-10 rat aortic
smooth muscle cells are obtained from the American Type
Culture Collection and are cultured in BME containing 10% FBS
and 50~g/mL gentamycin sulfate. Human ovarian carcinoma
cells (SKOV3) and a sub-line which has been selected fro
resistance to vinblastine (SKVLBl) were a generous gift from
Dr. Victor Ling of the Ontario Cancer Institute. Both cell
lines are maintained in BME cont~ining 10% FBS and 50~g/mL
gentamycin sulfate. Vinblastine is added to a final
concentration of l~g/mL to SKVLsl cells 24 hours after
passage to maintain selection pressure for P-glycoprotein-
overexpressing cells.
Cell Proliferation ~nd Cvcle Arrest Assavs
Cell proliferation assays are performed as described by
Skehan et al. For Jurkat cells, cultures are treated with the
indicated drugs as described in Skehan and total cell numbers
are determined by counting the cells in a hemacytometer. The
percentage of cells in mitosis are determined by staining
with 0.4% Giemsa in PsS followed by rapid washes with PsS.
At least 1000 cells per treatment are scored for the presence
of mitotic figures and the mitotic index is calculated as the
ration of the cells with mitotic figures to the total number
of cells counted.
Tmmunofluorescence Assavs
A-10 cells are grown to near-confluency on glass
coverslips in BME/10% FBS. Compounds in PBS are added to the
indicated final concentrations and cells are incubated for an
additional 24 hours. For the staining of microtubules and
intermediate filaments, the cells are fixed with cold
methanol and incubated with PsS containing 10% calf serum to
block nonspecific binding sites. Cells are then incubated at
37'C for 60 min. with either monoclonal anti-~-tubulin or
with monoclonal anti-vimentin at dilutions recommended by the
SUBSTITUTE SHEET (RULE 26)
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manufacturer. Bound primary antibodies are subse~uently
visualized by a 45-minute incubation with fluorescein-
conjugated rabbit antimouse IgG. The coverslips are mounted
on microscope slides and the fluorescence patterns are
examined and photographed using a Zeiss Photomicroscope Ill
e~uipped with epifluorescence optics for fluorescein. For
staining of microfilaments, cells are fixed with 3%
paraformaldehyde, permeabilized with 0.2~ Triton X-100 and
chemically reduced with sodium borohydride (lmg/ML). PBS
containing lOOnM TRITC-phalloidin is then added and the
mixture is allowed to incubate for 45 min. at 3 7 C. The
cells are washed rapidly with PBS before the coverslips are
mounted and immediately photographed as described above.
Fffects of crv~to~hvcins and vinblastine on Jurkat cell
~roli~eration and cell cvcle
Dose-response curves for the effects of cryptophycin
compounds and vinblastine on cell proliferation and the
percentage of cells in mitosis are determined.
Effects of cvtochalasin B, vinblastine and crv~to~hvcins on
the cvtoskeleton
Aortic smooth muscle (A-10) cells are grown on glass
coverslips and treated with PBS, 2~M cytochalasin B, lOOnM
vinblastine or lOnM cryptophycin compounds . After 24 hours,
microtubules and vimentin intermediate filaments are
visualized by indirect immunofluorescence and microfilaments
are stained using TRITC - phalloidin. The morphological
effects of each drug is examined. Untreated cells displayed
extensive microtubule networks complete with perinuclear
microtubule organizing centers. Vimentin intermediate
filaments were also evenly distributed throughout the
cytoplasm, while bundles of microfilaments were concentrated
along the major axis of the cell. Cytochalasin B caused
complete depolymerization of microfilaments along with the
accumulation of paracrystalline remnants. This compound did
not affect the distribution of either microtubules or
SUBSTITUTE SHEET (RULE 26)
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intermediate filaments. The cryptophycin treated
microtubules and vimentin intermediates are observed for
depletion of microtubules, and collapse of rimentin
intermediate filaments.
Effects of crv~to~hYcins and vinblastine on taxol-stabilized
microtubules
A-10 cells are treated for 3 hours with 0 or 10~M taxol
before the addition of PBS, 100nM vinblastine or 10nM
cryptophycin compound. After 24 hours, microtubule
organization is examined by ;mmnnofluorescence as described
above. Compared with those in control cells, microtubules in
taxol-treated cells were extensively bundled, especially in
the cell polar regions. As before, vinblastine caused
complete depolymerization o~ microtubules non-pretreated
cells. However, pretreatment with taxol prevented
microtubule depolymerization in response to vinblastine.
Similarly, microtubules pretreated with taxol are observed
with cryptophycin treatment.
~eversibilitv of microtubule de~olvmerization bv vinblastine
and crv~to~hvcin
A-10 cells are treated with either 100nM vinblastine or
10nM cryptophycins for 24 hr., resulting in complete
microtubule depolymerization. The cells are then washed and
incubated in drug-free medium for periods of 1 hour or 24
hours. Microtubules repolymerized rapidly after the removal
of vinblastine, showing significant levels of microtubules
after 1 hour and complete morphological recovery bv 24 hour.
Cells are visualized for microtubule state after treatment
with a cryptophycin compound of this invention at either 1
hour or 24 hours after removal of the cryptophycin compounds.
Effects of combinations of vinblastine and crv~to~hvcins on
cel 1 ~roliferation
SKOV3 cells are treated with combinations of
cryptophycins and vinblastine for 48 hours. The percentages
SUBSTITUTE SHEET (RULE 26)
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of surviving cells are then determined and the ICsoS for each
combination is calculated.
Toxic;tv of crv~to~hvcins, vinblastine and taxol toward SKOV3
and SKVTRl cells
SKVLBl cells are resistant to natural product anticancer
drugs because of their over expression of P-glycoprotein.
The abilities of taxol, vinblastine and cryptophycin
compounds to inhibit the growth of SKOV3 and SKVLBl cells are
observed. Taxol caused dose-dependent inhibition of the
proliferation of both cell lines with ICsos for SKOV3 and
SKVLBl cells of 1 and 8000nM, respectively Vinblastine also
inhibited the growth of both cell lines, with ICsoS of 0. 35
and 4200nM for SKOV3 and SKVLBl cells, respectively.
Cryptophycins compounds of this invention demonstrate
activity with an ICsoS of from about 1 to about lOOOpm for
SKOV3 and SKVLBl cells.
Thus it can be demonstrated that the present invention
provides novel cryptophycin compounds which are potent
inhibitors of cell proliferation, acting by disruption of the
microtubule network and inhibition o~ mitosis. These studies
can illustrate that cryptophycin compounds disrupt
microtubule organization and thus normal cellular functions,
including those of mitosis.
Classic anti-microtubule agents, such as colchicine and
vinca alkaloids, arrest cell division at mitosis. It seems
appropriate to compare the effect of one of these agents on
cell proliferation with the cryptophycin compounds. For this
purpose, the vinca alkaloid vinblastine was selected as
representative of the classic anti-microtubule agents.
Accordingly, the effect o~ cryptophycin compounds and
vinblastine on the proliferation and cell cycle progression
of the Jurkat T-cell leukemia cell line is compared.
Since antimitotic effects are commonly mediated by
disruption of microtubules in the mitotic spindles, the
effects of cryptophycin compounds on cytoskeletal structures
are characterized by fluorescence microscopy.
SUBSTITUTE SHEET (RULE 26)
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Immunofluorescence st~;n;ng of cells treated with either a
cryptophycin compound or vinblastine demonstrate that both
compounds cause the complete loss of microtubules. Similar
studies with SKOV3 cells can show that the anti-microtubule
effects of cryptophycin compounds are not unique to the
smooth muscle cell line.
GC3 human Colon Carcinoma Screen
Selected wells of a 96 well plate were seeded with
GC3 human colon carcinoma cells (lx10 cells in 100~1 assay
medium/well) twenty four hours prior to test compound
addition Cell free assay medium was added to other select
wells of the 96 well plate. The assay medium (RPMI-1640 was
the medium used; however, any medium that will allow the
cells to survive would be acceptable) was supplemented with
10% dialyzed fetal bovine serum and 25 mM HEPES buffer.
The test compound was stored in an amber bottle
prior to testing. Fresh dimethylsulfoxide stock solution
(200~g/ml) was prepared immediately prior to preparation of
test sample dilutions in phosphate-bu~fered saline (PBS). A
dilution of 1:20 dimethylsulfoxide solution in PBS was
prepared such that the final concentration was 10 ~g/ml.
Serial 1:3 dilutions using PBS (. 5ml previous sample of lml
PBS) were prepared. Falcon 2054 tubes were used for the
assay.
A 10ul sample of each dilution of test compound was
added in triplicate to wells of GC3 plates. The plates were
incubated for 72 hours at about 37 C. A 10 ~1 sample of
stock 3-[4,5-dimethyl-2-yl]-2,5-diphenyltetrazolium bromide
salt ("MTT" 5 mg/ml in PBS) was added to each well. The
plates were incubated for about an hour at 37 C. The plates
were centri~uged, media was decanted from the wells and 100~1
acid-isopropanol (0.04 N HCl in isopropanol) was added to
each well The plate was read within one hour using a test
wavelength of 570nm (SpectraMax reader).
Evaluation of compounds of Formula I suggest that
the compounds can be useful in the treatment methods claimed
S~,~ 111 ~JTE SHEET (RULE 26)
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-34-
herein. Further, the compounds will be useful for disrupting
the microtubule system.
Compounds of Formula I can be prepared using a
compound of the formula II
R1 R3 R5
Ar~\R O
o~/X oR24
R9--~X"---NHR13
R1 o R7 R8
II
wherein
Ar Rl R2 R3 R4, R5, R7, R8, R9, Rl0 have the meanings set
for supra in Formula I.
Rl3 is selected from the group consisting of t-butylcarbamate
(sOC);
R24 is selected from the group consisting of
\N~5
(N-hydroxysuccinimide, herein "NHS"), N-
hydroxysulfosuccinimide and salts thereof, 2-nitrophenyl, 4-
nitrophenyl, and 2,4-dichlorophenyl;
X is O, NH or alkylamino;
Y is O, NH, or alkylamino.
Compounds of Formula III
SUBSTITUTE SHEET (RULE 26)
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R1 R3 R5
Ar/~\R O
o~/X HN _r R6
O Co2H
3 11
R10 ~--X'''--' N HR1 3
R7 R8 III
wherein the R groups and various substituents are as defined
hereinbefore and throughout the specification; can be
prepared by contacting a compound of the formula IV
R1 R3 R5
Ar~/~O
o~/X oR25
R9 ~J~ NHR13
R1o ~\
R7 R8 IV
R25 is an active ester substituent;
with an acid of the formula
\~R6
Co2R27
R27 is selected from the group consisting of H, C1-C12 alkyl,
and aryl;
and a silylating agent. Bis N,O-trimethylsilyl acetamide
(BSA) is an especially preferred silylating agent.
As used with regard to R25 the phrase "active ester
substituent~ refers to a substituent which makes the oR24
substituent a good leaving group. Appropriate substituents
can be selected with guidance from standard reference guides,
for example, ~Protective Groups in Organic Chemistry~, Plenum
Press, (London and New York, 1973); Greene, T.W. ~Protecting
SUBSTITUTE SHEET (RULE 26)
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Groups in Organic Synthesis", Wiley (New York, 1981). An
especially pre~erred R25 group is N-hydroxy-succinimide. (NHS)
The processes described herein are most preferably
completed in the presence of a solvent. The artisan can
select an appropriate solvent for the above described
process. Inert organic solvents are particularly preferred;
however, under certain conditions an a~ueous solvent can be
appropriate. For example, if R27 is hydrogen and and Rl3 is
BOC an a~ueous base as solvent will be effective.
When the desired R6 substituent in the compound of
Formula I contains an amine, then the amine substituent of
the R6 group must be protected using an amino protecting
group. The artisan can readily select an appropriate amino
protecting group using guidance from standard works,
including, for example, "Protective Groups in Organic
Chemistryll, Plenum Press, (London and New York, 1973);
Greene, T.W. ~Protecting Groups in Organic Synthesis", Wiley
(New York, 1981).
R27 should be a group that allows for the removal
of the -C02R27 substituent using acidic, neutral, or mild
basic conditions. Preferred R27 groups include, but are in no
way limited to, hydrogen, Cl-C6 alkyl, tricholoromethyl,
trichloroethyl, and methylthiomethyl. It is especially
preferred that R27 is hydrogen.
To provide further guidance for the artisan, the
following schemes are provided:
SUBSTITUTE SHEET (RULE 26)
CA 02230540 1998-02-26
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-37-
CH3 Scheme I CH3
,0lN a4 LiOH ~ ~ O
- MeO Ace~ne - OH
OTBS OTBS
EDC,NHS,D~DF
H3C
. 50% aq HF
OH ~ CH3CN SBTO O ~ N,
O O
Scheme I'
Rl' R3 R5 R3
Ar ~ ~ 0~ ~ Ar ~
~ i ~ , Ar ~ ;b
As used in Scheme I' and throughout the
specification, Rl is halogen, SH, amino, monoalkylamino,
dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium,
sul~ate, phosphate or a protected OH or protected SH group;
R2 is OH or SH; R26 is an alcohol protecting group introduced
during a portion of the synthetic process to protect an
alcohol group which might otherwise react in the course o~
chemical manipulations, and is then removed at a later stage
of the synthesis. Numerous reactions for the formation and
51,~S ~ )TE SHEET (RULE 26)
CA 02230540 l998-02-26
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removal of such a protecting groups are described in a number
of standard works, including, for example, "Protective Groups
in Organic Chemistry~, Plenum Press, (London and New York,
1973); Greene, T.W. "Protecting Groups in Organic Synthesis",
Wiley (New York, 1981). The skilled artisan can select an
appropriate alcohol protecting group particularly with
guidance provided from such works. One particularly useful
alcohol protecting group is tert-butyldimethylsilyl (TBS).
COzl~
CH3 ~OJ~NHBoc
O J~
OH ONHS (iv)
CH3
CH3 ~_~ O
~O 1~1 o ONHS
~X~ HN~,R6 (v) ,~~~CNHB~c
J~ ~J~cNHBoc
CH3
~0
( -Vli) W o~,O H XR6
,~--~~ C NH ~
R6 has the meaning defined supra .
SUBSTITUTE SHEET (RULE 26)
CA 02230540 1998-02-26
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-39-
CH3
X R6
H
mcpba
o CH3
~0 ~
o~ H XR6
J~'~~C NH ~
TMSCVseparate
Cl CH3 ~ \ Cl CH3
=~R6 O~R6
,~~~C NH ~ ,~~J~C H ~
base
base
~0 ~0
o~ H ~R6 o~/ H l~R6
~J~C H ~ ~~J~C H ~
SUBSTITUTE SHEET (RULE 26)
CA 02230540 1998-02-26
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--~0--
The product o~ the schemes provided herein can be
further derivatized using standard methods to provide further
cryptophycin compounds.
The artisan can utilize appropriate starting
materials and reagents to prepare desired compounds using the
guidance of the previous schemes and following examples.
The ester starting material can be prepared, for
example, as follows:
~~CHO
St~p 1
HEW
TMG
~, CO2MQ
SIOP 2
DIBAL
OH
SteP 3
SAE
OH
Me3AI
MQ
OH
~J OH Step 5
BU2Sn=O
TsCI, TEA
SI~S ~ JTE SHEET (RULE 26)
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-41-
Me
~~~I~OTs
OH
Step 6
TBS-OTf
Et3N
Me
OTs
OTBS
Step 7
NBS/AIBN
Br Me
~OTs
OTBS
Step 8
DB U/ACN
Me
~~OTs
OTBS
Step 9
KCN
Me
--CN
OTBS
Step 10
DIBAL;
HEW
Me
~0
~J OTBS OMe
R6 has the meaning defined supra .
SUBSTITUTE SHEET (RULE 26)
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-42-
The scheme ~or preparing the ester is further
explained by the Preparation Section herein which provides
one speci~ic application of the scheme for the convenience of
the skilled artisan.
The Scheme for preparing the ester is applicable to
the Ar substituents claimed herein. The scheme illustration
is not intended to limited the synthesis scheme only to the
phenyl ring illustrated. Rather, the artisan can broadly
apply this process to provide desired starting materials for
the compounds claimed herein.
The necessary reaction time is related to the
starting materials and operating temperature. The optimum
reaction time for a given process is, as always, a compromise
which is determined by considering the competing goals of
throughput, which is favored by short reaction times, and
maximum yield, which is ~avored by long reaction times.
To further illustrate the invention the following
examples are provided. The scope o~ the invention is in no
way to be construed as limited to or by the ~ollowing
examples.
Pre~aration
Step 1. Methyl 5-Phenylpent-2 (E) -enoate. A solution of
trimethyl phosphonoacetate (376 g, 417 mL, 2.07 mol) in THF
(750 mL) was stirred at 0 ~C in a 3L 3-neck round bottom
flask equipped with a mechanical stirrer and M2 inlet. To
the chilled solution, neat tetramethyl guanidine (239 g, 260
mL, 2.07 mol) was added dropwise via an addition funnel. The
chilled clear pale yellow solution was stirred for 25 minutes
at 0 ~C. A solution of hydroc;nn~m~ldehyde (90%, 253 g, 248
mL, 1.9 mol) in THF (125 mL) was added dropwise to the
reaction solution slowly. Upon completion o~ addition, the
reaction was stirred for 10 h rising to room temperature. GC
indicated a 95:5 ratio of product to starting material.
500ml of water was added to the reaction vessel and the
reaction stirred overnight separating into two layers. The
SUBSTITUTE SHEET (RULE 26)
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organic layer was isolated and the aqueous layer was
extracted with t-BuOMe. The organic layers were combined and
dried over MgSO4, then concentrated in vacuo to yield an
orange oil. The crude product was distilled at 129 ~C/0.3mm
Hg yielding 360.5g, 91.7% yield, of a clear slightly yellow
oil.
EIMS m/z 190(13; M+), 159(410, 158(39), 131(90), 130(62),
117(22), 104(12), 95(57), 91(100), 77(21), 65(59); HREIMS m/z
190.0998 (C12H14O2 D -0.4 mnu); W lmax (e) 210 (8400), 260
(230) nm; IR nmax 3027, 2949, 1723, 1658, 1454, 1319, 1203,
978, 700 cm~l; 1H NMR d (CDCl3) 7.15-7.3 (Ph-H5;bm), 7.00
(3-H;dt, 15.6/6.6), 5.84 (2-H;dt, 15.6/1.2), 3.70 (OMe;s),
2.76 (5-H2;t, 7.2), 2.51 (4-H2; bdt, 6.6/7.2); 13C NMR d
(CDCl3) 166.9 (1), 148.3(3), 140.6(Ph-1'), 128.4/128.2
(Ph2'/3'/5'6'), 126.1 (Ph 4'), 121.4 (2). 51.3 (OMe),
34.2/33.8 (4/5).
Step 2. 5-phenyl-pent-2-en-1-ol. To a 12L 4-neck round
bottom ~1ask equipped with a thermocouple, mechanical stirrer
and N2 inlet, a solution o~ enoate ester (310 5 g, 1.5 mol)
in THF (1.5 L) was charged and chilled to -71 ~C via a i-
PrOH/CO2 bath. To the reaction vessel, was added dropwise
DIsAL (2.5 L, 1.5 M in toluene, 3.75 mol) at a rate to
maintain the reaction temperature < -50 ~C. Upon complete
addition, the reaction was stirred overnight with the
reaction temperature < -50 ~C. TLC (3:1 Hexanes:EtOAc, SiO
indicated absence of starting material a~ter 16 h. The
reaction temperature was allowed to raise to -15~C. The
reaction was quenched slowly withlN HCl (150 mL). At this
point the reaction setup into a gelatinous solid. A spatula
was employed to breakup the the semi-solid and lN HCl (200
mL) was added making the mixture more ~luid. Concentrated
HCl (625 mL) was charged to ~orm a two phase system. The
layers were separated and the product extracted with t-BuOMe.
The organic layer was dried over MgSO4 and concentrated in
vacuo to yield a clear pale yellow oil, 247.8g. The crude
SUBSTITUTE SHEET (RULE 26)
CA 02230~40 1998-02-26
W O 97/07798 PCT~US96/13855
product was distilled at la5 ~C/0.25mm Hg yielding 209.7g,
86.2%.
EIMS m/z 162 (l:M+) 144 (16), 129 (7), 117 (9) 108 (6), 92
(17), 91 (100), 75 (5), 65 (12), HREIMS m/z 162, 1049
(C11H14O, D -0.4 mmu); W lmax (e) 206 (9900), 260 (360); IR
nmax 3356, 2924, 1603, 1496, 1454, 970, 746, 700 cm~l; lH NMR
d 7.15-7.3 (Ph-H5;m), 5.70 (3-H;dt, 15.6/6.0), 5.61 (2-H;dt,
15.6/4.8), 4.02 (1-H2;d 4.8), 2.68 (5-H2; t, 7.2), 2.40
(OH;bs), 2.36(4-H2; dt, 6.0/7.2); 13C NMR dl41.6 (Ph 1'),
131.8(3), 129.5 (2), 128.3/128.2 (Ph 2'/3'/5'/6'), 125.7 (Ph
4'), 63.3 (1), 35.4/33.8 (4/5).
Step 3. (2S,3S)-2,3-Epoxy-5-phenyl-l-l~>entanol. To a
lL 3 neck round bottom flask e~uipped with a mechanical
stirrer, thermocouple and nitrogen inlet was added CH2Cl2 (350
mL), dried 4 A molecular sieves (30 g) and L-(+)-diethyl
tartrate (7.62 g, 0.037 mol). The resulting mixture was
cooled to -20 ~C and treated with Ti(O-i-Pr)4 (9.2 mL, 0.031
mol), followed by the addition of t-butylhydroperoxide (4.0 M
in CH2C12, 182 mL, 0.78 mol) at a rate to maintain the
temperature < -20 ~C. Upon complete addition, the reaction
mixture was stirred for another 30 min, and then treated with
a solution of the allylic alcohol (50 g, 0.31 mol) in CH2C12
(30 mL) at a rate to maintain the temperature S -20 ~C. The
reaction was stirred at the same temperature for 5 h, then
filtered into a solution of ferrous sulfate heptahydrate (132
g) and tartaric acid (40 g) in water (400 mL) at 0 ~C. The
mixture was stirred for 20 min, then transferred to a
separatory funnel and extracted with t-BuOMe (2x200 mL). The
combined organic phase was stirred with 30% NaOH solution
containing NaCl, for 1 h at 0 ~C. The layers were again
separated, and the aqueous phase extracted with t-BuOMe. The
combined organic phase was washed with brine, dried over
MgSO4 and concentrated to yield 52.8 g as an amber oil.
Step 4. (2R, 3R)-2-hydroxy-3-methyl-5-phenylpentan-1-
ol . To a 5L 3 neck round bottom flask equipped with a
mechanical stirrer, thermocouple and nitrogen inlet was added
hexanes (lL) and cooled to 0 ~C. A 2.0M solution of Me3Al in
SUBSTITUTE SHEET (RULE 26)
CA 02230~40 l998-02-26
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-45-
hexanes (800 mL, 1.6 mol) was added, followed by a solution
of the epoxide (120 g, 0.677 mol) in hexanes (250 mL)/CH2Cl2
(50 mL) maintaining the temperature below 20 ~C. Upon
complete addition, the cloudy reaction mixture was stirred at
5 ~C for 35 min, whereupon a solution of 10% HCl (300 mL) was
added dropwise, followed by the addition of concd HCl (350
mL). The layers were separated, and the organic phase was
washed with brine and dried over MgSO4. After removal of the
volatiles in vacuo, 122.1 gram of an oil was obtained.
Step 5. (2R, 3R)-2-hydroxy-3-methyl-5-phenylpent-1-yl
Tosylate. To a 2L 3 neck round bottom flask equipped with a
mechanical stirrer and nitrogen inlet was added the diol (58
g, 0.30 mol), dibutyltin oxide (1.5 g, 0.006 mol, 2 mol%),
toluenesulfonyl chloride (57.5 g, 0.30 mol), CH2Cl2 (580 mL)
and triethylamine (42.0 mL, 0.30 mol). The resulting mixture
was stirred at room temperature for 2 h (although the
reaction was complete within 1 h), filtered, washed with
water and dried over MgSO4. Concentration of the volatiles
in vacuo afforded 104.1 gram of a slightly amber oil.
Step 6. (2R, 3R) -2- [ ( tert-Butyldimethyl~ilyl)oxy]-3-
methyl-5-phenylpent-1-yl Tosylate. A solution of the
tosylate (100 g, 0.29 mol) and triethylamine (81.0 mL, 0.58
mol) in CH2C12 (1200 mL) was treated with neat TBS-OTf (99 mL,
0.43 mol) dropwise with continued stirring for another 20
min. The reaction was washed twice with brine, dried over
MgSO4 and concentrated to dryness. The oil was dissolved in
a m;nim~l amount o~ hexanes and filtered over a silica pad,
eluting with hexanes:EtOAc (9:1) to yield a slightly amber
oil, 134 g.
Step 7. (2R, 3R, 5RS) -2-[(tert-
Butyldimethylsilyl)oxy]-3-methyl-5-bromo-5-phenylpent-
l-yl Tosylate. To a 5L 3 neck round bottom flask equipped
with a mechanical stirrer, reflux condenser and nitrogen
inlet was added CCl4 (1680 mL), TBS Ts (140 g, 0.30 mol), Nss
(65g, 0.365 mol) and AIBN (16.5~g, 0.10 mol). The mixture
was degassed by evacuation under full vacuum with stirring,
and back~illing with nitrogen (3x). The reaction mixture was
SUBSTITUTE SHEET (RULE 26)
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-46-
then heated to re~lux, whereupon the color became dark brown.
After 15 min at vigorous re~lux, the reaction mixture became
light yellow, and chromatographic analysis indicated the
reaction was complete. A~ter cooling to room temperature,
the reaction was filtered and the ~iltrate concentrated to
dryness. The residue was redissolved in hexanes and ~iltered
again, and concentrated to dryness to a~ord 170.3 gram as an
amber oil.
Step 8. (2R, 3R) -2- [ ( tert-Butyldimethylsilyl)oxy]-3-
methyl-S-phenylpent-4(E)-en-1-yl Tosylate. To a 2L 3
neck round bottom ~lask e~uipped with a mechanical stirrer,
re~lux condenser and nitrogen inlet was added a solution of
the bromide (100 g, 0.186 mol) in acetonitrile (700 mL). DBU
(83. 6 mL, O.557 mol) was added and the resulting dark brown
solution was stirred at re~lux ~or 15 min. After cooling to
room temperature, the solvent was removed in vacuo, and the
residue digested in CH2C12 (200 mL) and ~iltered through a
silica pad. The volatiles were again evaporated, and the
residue dissolved in EtOAc and washed with water, brine and
dried over MgSO4 and concentrated to dryness. Preparative
mplc (Prep 500) chromatography a~forded the desired
unsaturated compound (50.3 g, 60% yield over 4 steps).
Step 9. (3S, 4R)-3-t(tert-Butyldimethylsilyl)oxy]-4-
methyl-6-phenylhex-5(E)-en-1-nitrile. The tosylate (50
g, 0.11 mol) was dissolved in DMSO (1 L) and treated with KCN
(14.2 g, 0.22 mol) and water (25 mL), and the resulting
mixture was stirred at 60 ~C under nitrogen ~or 18 h. A~ter
cooling to room temperature, the reaction mixture was
partitioned between EtOAc 11 L) and water (1 L). The
aqueous phase was extracted with EtOAc (500 mL), and the
combined organic phase was washed with brine and dried over
Na2SO~. Flash chromatography over silica with CH2Cl2 a~orded
the desired nitrile in 92% yield.
~tep 10. Methyl (5S, 6R)-5-t(tert-
Butylaimethylsilyl)oxy]-6-methyl-8-phenylocta-
2(E),7(E)-dienoate. The nitrile (14.67 g, 46.5 mmol) was
dissolved in toluene (200 mL) and cooled to -78 ~C under
SUBSTITUTE SHEET(RULE 26)
CA 02230~40 1998-02-26
W O 97/07798 PCTrUS96/13855
-47-
nitrogen. A 1.5M solution of DIBAL in toluene (37.2 mL, 55.8
mmol) was added dropwise with vigorous stirring. Upon
complete addition, the cooling bath was removed and the
reaction was stirred at room temperature for 1 h. The
reaction mixture was carefully poured into lN HCl and the
mixture stirred at room temperature for 30 min. The layers
were separated, and the organic phase was washed with a
saturated a~ueous solution of sodium potassium tartrate (2x),
brine and dried over Na2SO4. The volatiles were removed in
10 vacuo, and the crude pale yellow oil was used directly in the
subsequent condensation.
The crude aldehyde from above was dissolved in THF (90 mL)
and treated with trimethyl phosphonoacetate (9.03 mL, 55.8
mmol) and tetramethylguanidine (7.0 mL, 55.8 mmol) at room
temperature under nitrogen. The reaction mixture was stirred
for 16 h, then partitioned between EtOAc (200 mL) and water
(100 mL). The a~ueous phase was back extracted with EtOAc
(100 mL), and the combined organic phase was washed with
water, brine and dried over Na2SO4. The volatiles were
removed in vacuo, and the crude yellow oil (17.0 g) was
chromatographed over silica gel with CH2Cl2 : cyclohexane (1 :
1 to 2 : 1) to afford 13.67 grams of the desired ester,
78.5%.
Preparation 2
CH3
~~
~ OTBS OH
Methyl ester (2.673 mmol) was dissolved in acetone and then
lN a~ueous LioH (26mL) added at room temperature. The cloudy
mixture was further diluted with acetone (20mL) and the
resulting yellow mixture stirred at room temperature for
23.5h. The reaction was diluted with diethylether (400mL) and
the organics washed with lN HCl (120mL), brine (200mL) and
H2O (160mL). The organics were dried and concentrated in
Sl~ 111 ~ITE SHEET (RULE 26)
CA 02230~40 1998-02-26
W O 97/07798 PCTAUS96/13855
-48-
vacuo to leave a yellow oil which was purified by column
chromatography (gradient: 5% AcOH + 20%-40% EtOAc/Hexanes )
to give carboxylic acid as a yellow oil (960mg, 100%).
1H NMR (CDCl3) d 7.38-7.19 (m,PhH5), 7.09 (ddd,J=15.2,7.6
and7.9 Hz,3-H), 6.38 (d,J=16 Hz,8-H), 6.16 (dd,J=16 and8 Hz,
7-H), 5.85 (d,J=15.8Hz,2-H),3.81-3.75 (m,5-H), 2.49-2.37
(m,6-H,4-CH2), 1.12 (d,J=6.7Hz,6-Me), 0.91 (s,SiCMe3), 0.065
(s,SiMe), 0.068 (s,SiMe) ppm;
IR u (CHCl3) 2957,2930,2858,1697,1258,1098,838 cm~l;
MS (FD) 360.2 (M+,100);
[a]D+87.6~ (c 10.5, CHC13);
Anal. calcd. for C21H32O3 re~uires: C,69.95; H,8.95%. Found:
C,69.19; H,8.39%.
SUBSTITUTE SHEET (RULE 26)
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-49-
Pre~ar~tion 3
CH3
~'r } O
~ ~ OTBS O - N ~
To a stirred solution of carboxylic acid (2mmol) in dry
dimethylformamide (5.50mL) was added 1-ethyl-3-(3-
dimethyaminopropyl)carbodiimide (2.4mmol) and M-
hydroxysuccinimide (2.6mmol) at room temperature. The mixture
was stirred for 28h and then diluted with EtOAc (100mL) and
washed with lN aqueous HCl (2x50mL), H2O (75mL), dried and
concentrated in vacuo to leave an oil. Crude product was
purified by column chromatography (gradient: 5-30%
EtOAc/Hexanes) to give active ester as a pale yellow oil
(724mg,80%).
lH NMR (CDCl3) d 7.36-7.20 (m,PhH5,3-H), 6.38 (d,J=16Hz,8-H),
6.14 (dd,J=16.1 and 8.0 Hz,7-H). 6.03 (d,J=16Hz,2-H), 3.79
(~,J=4.3Hz,5-H), 2.94 (brs,CH2CH2), 2.58-2.42 (m,6-H,4-CH2),
1.10 (d,J=6.8Hz,6-Me), 0.90 (s,SiCMe3), 0.05 (s,SiMe2) ppm;
IR u (CHCl3)
2957,2931,2858,1772,1741,1648,1364,1254,1092,1069,838 cm~1;
MS (FD) 457 (M+,100);
[a]D +71.3~ (c 10.1, CHCl3);
Anal. calcd. for C25H3sNO5 requires: C,65.61;H,7.71;M,3.06%.
Found: C,65.51;H,7.56; N, 3.02%.
Pre~aration 4
CH3
~,~,~ ~ ~0
~ OH O -N ~
To a stirred solution of silyl ether (2.50g,5.47mmol) in
CH3CN (130mL) was added 48% aqueous HF (15mL) at 0C. The
SUBSTITUTESHEET(RULE26)
CA 02230~40 1998-02-26
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-50-
solution was stirred at 0 C for 0.75h and then at room
temperature for 4h. The reaction was diluted with
diethylether (300mL) and washed with H2O until the wash was
~pH7. Organics were dried (MgSO4) and concentrated in vacuo
to give a yellow residue which was recrystallized from Et20
to give alcohol as white crystals (1.46g,78%).
H NMR (CDCl3) d 7.41-7.20 (m,PhH5,3-H), 6.48 (d,J=16Hz,8-H),
6.15-6.07 (m,7-H,2-H), 3.71-3.65 (m,5-H), 2.83 (brs,CH2CH2),
2.60-2.33 (m,6-H,4-CH2),1.95 (brs, 5-OH), 1.14 (d,J=6.8Hz,6-
Me) ppm;
IR u (KBr)
3457,1804,1773,1735,1724,1209,1099,1067,1049,975,744,694 cm~
1 ;
W (EtOH) lmaX 250 (e =20535) nm;
MS (FD) 343.2 (M+,100);
[a] D --57.8~ (c 10.56, CHCl3);
Anal. calcd. for ClgH2lNO5S re~uires: C,66.46;H,6.16;N,4.08%.
Found: C,66.49; H,6.16; N, 4.07%.
Exam~le
A
CH3
~ r~O
= O--No~
D ~ o ~ NHBoc
C
To a suspension of carboxylic acid (1.28g, 3.87mmol), in dry
dichloromethane (6mL) was added EDC (742mg,3.87mmol) and D~AP
(73mg,0.60mmol) and the mixture stirred at room temperature
for 0.5h. A solution of alcohol (1.02g, 2.97mmol) in
dichlormethane (5.5mL) was added to the reaction mixture and
SUBSTITUTE SHEET (RULE 26)
CA 02230~40 1998-02-26
W O 97/07798 PCTrUS96/13855
stirred ~or a further 0.3h.The reaction was diluted with
CH2C12 (200mL) and washed with lN aq. HC1
(2x 50mL), sat. aq. NaHCO3 (2x 50mL), H20 (50mL). The organics
were dried (MgSO4) and concentrated in vacuo to leave an oily
residue, which was puri~ied by column chromatography
(gradient: 10-30% EtOAc/Hexanes) to give the desired ester as
a yellow solid (1.68g,79%).
H NMR (CDCl3) unit A d 7.35-7.20 (m,PhH5,3-H), 6.43
(d,J=15.8Hz,8-H), 6.12 (d,J=15.9Hz,2H), 5.99 (dd,J=8.5
andl5.8 Hz,7-H), 5.06-5.08 (m,5-H), 2.85 (brs,CH2CH2), 2.68-
2.61 (m,6-H,4-CH2), 1.13 (d,J=6.8Hz,6-Me); unit C d 5.31
(brt,NH),3.28-3 25 (m,3-CH2),1.43 (s,CMe3), 1.21 (s,2-Me),
1.19 (s,2-Me); unit D d 4.95 (dd,J=9.8 and 3.8Hz,2-H), 1.73-
1.64 (m,3-H,4-H), 1.59-1.49 (m,3-H'), 0.85 (d,J=6.4Hz,5-Me),
0.82 (d,J=6.4,4-Me) ppm;
IR u (KBr) 3400, 2975,1743,1367,1206,1126,1145,1068 cm~1;
MS (FD) 657 (M+,100);
[a]D+39.5~ (c 10.38, CHCl3);
Anal. calcd. ~or C35H48N2O1o re~uires: C,64.01;H,7.37;N,4.27%.
Found: C,64.19;H,7.27; N,4.52 %.
~mnle 2
A
CH3
B
r O cO2H
D ~ oJ~ NHBoc
C
To a stirred solution o~ active ester (150mg, 0.229mmol) in
dry DMF (2.5mL) was added N,O-Bis-(trimethylsilyl)acetamide
(282uL,1.143mmol) ~ollowed by ~-Hydroxy-phenylglycine
(57mg,0.343mmmol). The mixture was heated in a sealed tube
under N2 at 55 C ~or 20h. Reaction solution was diluted with
SUBSTITUTE SHEET (RULE 26)
CA 02230~40 1998-02-26
W O 97/07798 PCTAJS96/13855
EtOAc (180mL) and washed with lN aq. HCl (50mL),H20 (50mL),
brine (5OmL), dried (MgSO4) and concentrated in vacuo to give
a yellow solid. Purification of the crude solid by column
chromatography (gradient: 5-20% MeOH/CH2C12) provided amide
(122mg,75%).
H NMR (CD30D/CDC13) Unit A d 7.27-7.20 (m,PhHs), 6.75-6.69
(m,3-H), 6.43 (d,J=15.9Hz,8-H), 5.96 (d,J=15.7Hz,7-H), 5.93
(d,J=15.6Hz,2-H), 4.95-4.93 (m,5-H), 2.56-2.49 (m,6-H,4-CH2),
1.04 (d,J=6.8Hz,6-Me); Unit B d 7.16 (d,J=8.3Hz,ArH2), 6.66
(d,J=8.2Hz,ArH2), 5.62 (brt,NH)5.19-5.18 (m,2-H); Unit C d
3.15 (d,J=6.3Hz,3-CH2), 1.36 (s,CMe3), 1.11 (s,2-Me), 1.08
(s,2-Me); Unit D d 4.85 (dd,J=9.6 and 3.3Hz,2-H), 1.64-1.57
(m,3-H,4-H), 1.55-1.47 (m,3-H'), 0.76 (d, J=6.3Hz,5-Me), 0.73
(d,J=6.3Hz,4-Me) ppm ;
IR u (KBr) 3400,2972,1728,1672,1614,1515,1450,1416,1171,1147
cm~l;
MS (FAB) 610.6 ([MH2-Boc]+,100);
[a] D -19.9~ (C 6.53, MeOH).
~x;lmnle 3
CH3
O ~ o HN ~ ~ B
O ~ H ~
C
Boc amine as prepared by Example 2 (109mg,0.154mmol) was
dissolved in trfluoracetic acid (5mL,5mM) and stirred at room
temperature for 2h.The reaction was concentrated in vacuo and
dried under high vacuum to give the trifluoroacetate salt of
amine as a light brown foam. Crude amine salt (max.
0.154mmol) was dissolved in dry DMF (31mL) and
diisopropylethylamine (80uL,0.462mmol), followed by
SU~ ~ JTE SHEET (RULE 26)
CA 02230~40 1998-02-26
W O 97/07798 PCT~US96/1385S
-53-
pentafluorophenyl diphenyl -phosphinate (77mg,0.2mmol) added.
The resulting solution was stirred at room temperature under
dry N2 for 15h, concentrated in vacuo and the residue
purified by column chromatography (gradient: 1-4% MeOH/CH2Cl2)
to provide cryptophycin as a tan solid (54mg,59%).
H NMR (CDCl3) Unit A d 7.36-7.15 (m,PhHs), 6.79-6.69 (m,3-H),
6.54 (d,J=15.8,8-H), 5.98 (dd,J= 15.8 and 8.8 Hz,7-H), 5.06-
5.0 (m,5-H), 2.61-2.49 (m,6-H,4-H), 2.39-2.30 (m,3-H'), 1.10
(d,J=6.8Hz,6-Me); Unit B d 7.90 (dd,J=10 and 1.68Hz,OH), 7.65
(d,J=6.3Hz,NH), 7.10 (d,J=8.5,ArH2), 6.71 (d,J=8.4,ArH2), 5.28
(d,J=6.5Hz,2-H), ; Unit C d 3.55-3.47 (dd,J=13.3 and
lO.lHz,3-CH2), 3.00 (d,J=13.4Hz,NH) 1.19 (s,2-Me), 1.16 (s,2-
Me); Unit D d 4.90 (dd,J=10 and 3.5Hz,2-H), 1.66-1.54 (m,3-
H,4-H), 1.32-1.25 (m,3-H'), 0.67 (apparent t,J=7.lHz,5-Me,4-
Me) ppmi
IR u (KBr)
3418,3340,2960,1740,1713,16711514,1271,1198,1155,972 cm-l;
MS (FD) 590 (M+,lOO)i
[a]D+15.35~ (c 3.91, CHCl3).
~le 4
A
CH3
~0
r O HN~ B
,~ J~ H
C
Styrene prepared as described by Example 3 (42mg, 0.0712mmol)
was suspended in dry dichloromethane (2.2mL, 0.035mM) and
mCPsA (49mg, 0.285mmol) added in one portion at room
temperature. Dry tetrahydrofuran (0.3mL) was added to produce
SUBSTITUTE SHEET (RULE 26)
CA 02230~40 1998-02-26
W O 97/07798 PCTAJS96/13855
a homogeneous solution. The reaction was stirred under N2 at
room temperture for 21h and then diluted with further CH2Cl2
(15mL). Organics were washed with 10% aq. Na2S2Os (lOmL), sat.
aq. NaHCO3 (10mL), H2O (10mL), dried (MgSO4) and concentrated
in vacuo to give a yellow solid. Crude product was initially
purified by column chromatography (gradient: 1-5% MeOH/
CH2Cl2) to give a 1: 1.15 mixture of a:b C7-C8 epoxides as a
white solid (23mg, 54%).Reverse phase HPLC (column: 4.6x250mm
Kromsil C18; Eluent: 60% CH3CN/ H2O; Flow: 1.0mL/min; W :
220nm) separation of the a:b mixture provided a-epoxide
(2.3mg, t=13.7min) and ~-epoxide (5.8mg, t=12.1min) as white
solids.
~Yam~le 5
CH3
~ ~OH
~1 0 HN B
~ H
C
The above illustrated compound was prepared substantially as
described above using the procedures of Examples 1-4
a-Epoxide:
lH NMR (CDCl3)
Exam~le 6
B
~ J~ H
SUBSTITUTE SHEET (RULE 26)
CA 02230~40 1998-02-26
W O 97/07798 PCTrUS96/13855
-55-
The above illustrated compound was prepared substantially as
described above using the procedures o~ Examples 1-4
~-Epoxide:
lH NMR (CDC13) Unit A d 7.36-716 (m,PhHs), 6.70-6.79 (m,3-H),
5.91 (dd,J=15.5 and 5.18Hz,2-H) 5.23-5.18 (m,5-H), 3.75
(d,J=1.67Hz,8-H), 2.96 (dd,J=7.4 and 2.0Hz,7-H), 2.72-2.67
(m,4-H), 2.44-2.39 (m,4-H'), 1.81-1.88 (m,6-H), 1.13
(d,J=6.9,6-Me); Unit B d 7.66 (s,NH), 7.13 (d,J=8.5Hz,ArH2),
6.74 (d,J=8.5Hz,ArH2), 5.27 (s,2-H); Unit C d 7.66 (s,NH),
3.49 (dd,J=13.6 and lOHz,3-CH2), 1.20 (s,2-Me),1.18 (s,2-
Me); Unit D d 4.93 (dd,J=10 and 3.2Hz,2-H), 1.69-1.59 (m,3-
H,4-H), 1.30-1.22 (m,3-H'), 0.79 (d,J=6.2Hz,5-Me), 0.78
(d,J=6. 3HZ, 4-Me) ppm.
SlJ-~S 1 1 1 UTE SHEET (RULE 26)
