CA2214565C - New cryptophycins from synthesis - Google Patents

Abstract

Novel cryptophycin compounds are disclosed, together with methods of producing cryptophycins by total synthesis and methods for the use of such cryptophycins in pharmaceuticals to inhibit the proliferation of mammalian cells and to treat neoplasia.

Description

NEW CRYPTOPHYCINS FROM SYNTHESIS
This invention was made in part with U.S. Government support under Grant Nos.
CA12623 and CA53001 from The National Cancer Institute, Department of Health and Human Services. Accordingly, the U.S. Government may have certain rights in this invention.

Background of the Invention 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. Clinical experience in chemotherapy has demonstrated that new and more effective drugs are desirable to treat these diseases. Such 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 in a dynamic state of assembly and disassembly; that is, heterodimers of tubulin are polymerized to form microtubules, and microtubules are depolymerized to their constituent components. 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 microtubules that form the mitotic spindle. 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 chemotherapeutic agents in clinical use.
Such anti-mitotic agents or poisons 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 consist 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 promotes the polymerization of tubulin and thus stabilizes microtubule structures.

However, merely having activity as an antimitotic agent does not guarantee efficacy against a tumor cell, and certainly not a tumor cell which exhibits a drug-resistant phenotype. Vinca alkaloids such as vinblastine and vincristine are effective against neoplastic cells and tumors, yet they lack activity against some drug-resistant tumors and cells. One basis for a neoplastic cell displaying drug resistance (DR) or multiple-drug resistance (MDR) is through the over-expression of P-glycoprotein.
Compounds which are poor substrates for transport of P-glycoprotein should be useful in circumventing such DR or MDR phenotypes.
Accordingly, the exhibition of the DR or MDR phenotype by many tumor cells and the clinically proven mode of action of anti-microtubule agents against 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.
Agents which have shown promise in this regard include a class of compounds known as cryptophycins.
With respect to methods of producing cryptophycins, no method for total synthesis of cryptophycins is currently known. Cryptophycin compounds are presently produced via isolation from blue-green alga or are semi-synthetic variations of such naturally produced compounds. The lack of a total synthetic method necessarily makes it difficult to produce stereospecific cryptophycins which can maximize activity and increase the stability of the compound. For example, research has shown that cryptophycins with an intact macrocyclic ring are more active. Accordingly, a total synthetic method which could produce cryptophycins with a macrocyclic ring that is more stable than naturally derived cryptophycins would be desirable. The present invention solves these problems.

Disclosure of the Invention The present invention provides novel cryptophycin compounds having the following structure:

RiR2R3 ~R5 Ar X HN Rs Rio Y HN O
O g wherein Ar is methyl or phenyl or any simple unsubstituted or substituted aromatic or heteroaromatic group;
Rl is a halogen, SH, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, or phosphate;
R2 is OH or SH; or Rl and R2 may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring or a monoalkyiphosphate ring; or R, and R2 may be taken together to form a double bond between C18 and C19;
R3 is a lower alkyl group;
R4 and R5 are H; or R4 and R5 may be taken together to form a double bond between C13 and C14;
R6 is a benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, haloalkoxybenzyl, or dihaloalkoxybenzyl group;
R7, Rg, R9 and Rlo are each independently H or a lower alkyl group; and X and Y are each independently 0, NH or alkylamino.

- 3a -In a further aspect, the present invention provides a cryptophycin represented by the structure:

Rt R3 Rs R2 ~
Ar ,a 0 X Gl HN Rs ~ c ; ::C
Rio Y HN 0 O ~
R"t ~s wherein Ar is a phenyl group, a phenyl group substituted with a C1-C5 alkyl group, a phenyl group substituted with a Cl-C5 alkoxyl group, a phenyl group substituted with a halogen, a naphthyl group, a furyl group, an indolyl group, a pyrrolyl group, a pyridyl group, or a thienyl group;
Rl is a halogen, SH, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, or phosphate;
R2 is OH or SH; or Rl and R2 taken together form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring or a monoalkylphosphate ring; or R, and R2 taken together form a double bond between C18 and C19;
R3 is a C1-C5 alkyl group;
R4 and R5 are H; or R4 and R5 taken together form a double bond between C13 and C14;
R6 is a benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, haloalkoxybenzyl, or dihaloalkoxybenzyl group;
R7 and Rg are each independently a CI -C5 alkyl group;
R9 and Rlo are each independently H or a C1-C5 alkyl group; and -3b-X and Y are each independently 0, NH or alkylamino, wherein the following structures are excluded:

H GHs I Q ,yi O O HN G!

4 HN O OCHg t~) o HN cl fl (2) The invention further provides a pharmaceutical composition useful for inhibiting the proliferation of a hyperproliferative mammalian cell comprising an effective amount of a compound as described in the preceding paragraph together with a pharmaceutically acceptable carrier.

In a further aspect, the invention provides use of a cryptophycin as described above for the production of a medicament.

- 3c -In a further aspect, the invention provides use of a cryptophycin as described above for the treatment of a pathological condition caused by hyperproliferating mammalian cells.

The present invention further provides total synthetic methods for producing cryptophycins. The present invention also provides for the use of cryptophycins in pharmaceuticals, to inhibit the proliferation of mammalian cells and to treat neoplasia.
Brief Description of the Drawings Figure 1 provides a general structure of selected cryptophycin compounds of the present invention and a numbering system for the hydroxy acid units A and D
and amino acid units B and C in selected embodiments;
Figures 2A and B graphically depict the effects of cryptophycin compounds and vinblastine on Jurkat cell proliferation and cell cycle progression. Jurkat cells were incubated with the indicated concentrations of cryptophycin compounds (A) or vinblastine (B) for 24 hours. For each sample, the number of viable cells (~) and the mitotic index (0) were determined as described in the Experimental Section. Values represent the means standard deviation (sd) for triplicate samples in one of three similar experiments;
Figure 3 graphically depicts the reversibility of the effects of vinblastine, cryptophycins and taxol on cell growth. SKOV3 cells were treated with 0.1nM
vinblastine (0), 0.1nM cryptophycins (M) or 1nM taxol (19) at time = 0. These concentrations inhibited cell growth by 50% for each compound. After 24 hours the cells were washed and incubated in drug-free medium for the time indicated. The cell density was determined by sulforhodamine B (SRB) staining as described in the Experimental Section,. and is expressed as the mean sd absorbance at 560 run for triplicate samples in one of three experiments;
Figure 4 provides Isobolograms for combinational effects of vinblastine and cryptophycins on cell proliferation. SKOV3 cells were treated with vinblastine (0-600pM) and/or cryptophycins (1-100pM) for 48 hours. Cell numbers were then determined by SRB staining as described in the Experimental Section, and the IC50s (M) and the line of additivity (---) were determined for combinations of vinblastine and cryptophycin compounds. Values represent means for two experunents each containing triplicate samples;
Figure 5 provides a first scheme for synthesizing cryptophycins in accordance with the present invention;

Figure 6 provides a scheme for producing a hydroxy acid unit A;
Figure 7 provides a scheme for producing the subunit of a cryptophycin comprising a hydroxy acid unit A and amino acid B;
Figure 8 provides a scheme for producing the subunit of a cryptophycin comprising an amino acid unit C and hydroxy acid D;
Figure 9 provides a first scheme for synthesizing selected cryptophycins in accordance with the present invention;
Figure 10 provides a second scheme for synthesizing selected cryptophycins in accordance with the present invention;
Figure 11 provides a scheme for synthesizing a subunit of a cryptophycin comprising a hydroxy acid D;
Figure 12 provides a third scheme for synthesizing selected cryptophycins in accordance with the present invention;
Figure 13 provides a fourth scheme for synthesizing selected cryptophycins in accordance with the present invention; and Figure 14 provides a fifth scheme for synthesizing selected cryptophycins in accordance with the present invention.

Detailed Description of the Invention The present invention provides novel cryptophycin compounds having the following structure:

Ri ~
R2 ~Rs Ar ,3 O
,8 ,s ,4 X HN Rs Rs s Rio Y HN O
O s wherein Ar is methyl or phenyl or any simple unsubstituted or substituted aromatic or heteroaromatic group;
Rl is a halogen, SH, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, or phosphate;
R2 is OH or SH; or R, and R2 may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring or a monoalkyiphosphate ring; or Rl and R2 may be taken together to form a double bond between C18 and C19;
R3 is a lower alkyl group;
R4andR5 are H;or R4 and R5 may be taken together to form a double bond between C13 and C14;
R6 is a benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, haloalkoxybenzyl, or dihaloalkoxybenzyl group;
R7, R8, R9 and Rlo are each independently H or a lower alkyl group; and X and Y are each independently 0, NH or alkylamino.

In one aspect of the present invention, novel cryptophycin compounds are provided having the following structure:

R1 R3 Rs 11 10 8 s 6 Wherein R, is H, OH, a halogen, 0 of a ketone group, NH2, SH, a lower alkoxyl group or a lower alkyl group;
R2 is H, OH, 0 of a ketone group, NH2, SH, a lower alkoxyl group or a lower alkyl group; or Rl and R2 may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring or a double bond between C,o and Cll; or Rl and R4 may be taken together to form a tetrahydrofuran ring;
R3 is H or a lower alkyl group;
R4 is OH, a lower alkanoyloxy group or a lower a-hydroxy alkanoyloxy group;
R5 is H or an OH group;
R6 is H; or R5 and R6 may be taken together to form a double bond between C5 and C6;
R7 is a benzyl, hydroxybenzyl, methoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, halomethoxybenzyl, or dihalomethoxybenzyl group;
R. is OH, a lower #-amino acid wherein Cl is bonded to N of the Q-amino acid, or an esterified lower 0-amino acid wherein Cl is bonded to N of the esterified lower 0-amino acid group;
R4 and R$ may be taken together to form a didepsipeptide group consisting of a lower a-amino acid bonded to a lower a-hydroxy alkanoic acid; and R5 and RS may be taken together to form a didepsipeptide group consisting of a lower a-amino acid bonded to a lower a-hydroxy alkanoic acid; and with the following provisos:
R, is H, a lower alkyl group, or a lower alkoxyl group only if R2 is OH, 0 of a ketone group, NHZ, SH;
R2 is H, a lower alkyl group, or a lower alkoxyl group only if Rl is OH, 0 of a ketone group, NH2, SH;
when R, is OH, R. is OH, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R4 and R8 are taken together to form the didepsipeptide group with the structure X:

O O O

X= 3 4 JL a a Rio 0 ' wherein O, of X corresponds to R4, N8 of X corresponds to R8,. R9 is methyl, and Rlo is isobutyl, R7 is not 3-chloro-4-methoxybenzyl;
when R, and R2 are taken together to form an epoxide ring, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R4 and R8 are taken together to form a didepsipeptide with the structure X, R9 is methyl, and Rlo is isobutyl, R7 is not 3-chloro-4-methoxybenzyl;
when R, and R2 are taken together to form a double bond between Cla and Cl1, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R4 and R8 are taken together to form a didepsipeptide with the structure X, R9 is methyl, and Rio is isobutyl, R7 is not 3-chloro-4-methoxybenzyl; and when Rl and R2 are taken together to form an epoxide group, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C61 R4 is bonded to the carboxy terminus of leucic acid, and R8 is bonded to the nitrogen terminus of either 3-amino-2-methylpropionic acid or 3-amino-2-methylpropionic acid methyl ester, R7 is not 3-chloro-4-methoxybenzyl.
The invention further provides cryptophycin compounds wherein at least one of the groups attached to C2, Cg, C9, C,o, and Cll has R stereochemistry. In a further embodiment of the invention, at least one of the groups attached to C2, C81 C91 C,o, and C11 has S stereochemistry.
The invention further provides cryptophycin compounds in accordance with the above structure where the structure of the didepsipeptide that is formed when R4 or R5 is taken together with R. is the following structure X:

O

X= J3 4 6 8 Ri0 :OS 7 H

wherein O1 of X corresponds to R4 or R5, N8 of X corresponds to R8, R9 is H or a lower alkyl group, and Rlo is H or a lower alkyl group.
As used herein, the following terms have the indicated meanings unless a contrary meaning is clearly intended from the use in context:
"lower Q-amino acid" means any Q-amino acid having three to eight carbons and includes linear and non-linear hydrocarbon chains; for example, 3-amino-2-methylpropionic acid.
"esterified lower (3-amino acid" means any 0-amino acid having three to eight carbons where the hydrogen of the carboxylic acid group is substituted with a methyl group; for example, 3-amino-2-methylpropionic acid methyl ester.
"lower alkanoyloxy group" means an alkanoyloxy group of one to seven carbons and includes linear and non-linear hydrocarbon chains.
"lower a-hydroxyalkanoyloxy group" means an a-hydroxyalkanoyloxy group of two to seven carbons and includes linear and non-linear hydrocarbon chains;
for example, 2-hydroxy-4-methylvaleric acid.
"lower alkoxyl group" means any alkyl group of one to five carbons bonded to an oxygen atom.
"lower alkyl group" means an alkyl group of one to five carbons and includes linear and non-linear hydrocarbon chains including, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, methylated butyl groups, pentyl, and tert-pentyl groups.
"allylically substituted alkene" means any alkene which contains an alkyl substitution.
"epoxide ring" means a three-membered ring whose backbone consists of two carbons and an oxygen atom.
"aziridine ring" means a three-membered ring whose backbone consists of two carbons and a nitrogen atom.
"episulfide ring" means a three-membered ring whose backbone consists of two carbons and a sulfur atom.
"sulfate ring" means a five-membered ring consisting of a carbon-carbon-oxygen-sulfur-oxygen backbone with two additional oxygen atoms connected to the sulfur atom.
"monoalkyiphosphate ring" means a five-membered ring consisting of a carbon-carbon-oxygen-phosphorus-oxygen backbone with two additional oxygen atoms, one of which bears a lower alkyl group, connected to the phosphorus atom.
"simple unsubstituted aromatic group" refers to common aromatic rings having 4n+2 pi electrons in a monocyclic conjugated system (for example, furyl, pyrrolyl, thienyl, pyridyl) or a bicyclic conjugated system (for example, indolyl or naphthyl).
"simple substituted aromatic group" refers to a phenyl group substituted with single group (e.g. a lower alkyl group or a halogen).
"heteroaromatic group" refers to aromatic rings which contain one or more non-carbon substituents such as oxygen, nitrogen, or sulfur.
"halogen" refers to those members of the group on the periodic table historically known as the 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 those of ordinary skill in the art.l=2 One embodiment of a cryptophycin compound of the present invention is when R, and R2 are taken together to form an epoxide group, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 4-methoxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is methyl and Rlo is isobutyl. The structure of this compound, Cryptophycin 2, is as follows:

O
O =
li O-_ O O HN
NO
~Ha A further embodiment of a compound of the present invention is when Rl and R2 are taken together to form a double bond between the C,o and Cll carbons, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 4-methoxybenzyl, and R4 and R. are taken together to form the didepsipeptide with the structure X where R9 is methyl and R,o is isobutyl. The structure of this cryptophycin compound, Cryptophycin 4, is as follows:
CJi5iTO
O" 00 HN
o H ~o OOFIs ~

A further embodiment of a compound of the present invention is when R, and R4 are taken together to form a tetrahydrofuran ring, R2 is an OH group, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-4-methoxybenzyl, and R8 is a (2-carbomethoxypropyl)amino group. The structure of this compound, Cryptophycin 6, is as follows:

HN CI

A further embodiment of a compound of the present invention is when Rl and R4 are taken together to form a tetrahydrofuran ring, R2 and R8 are OH groups, R3 is methyl, R5 and R6 are taken together to form a double bond between C. and C6 such that there is a double bond, and R7 is 3-chloro-4-methoxybenzyl. The structure of this compound, Cryptophycin 7, is as follows:

I ~ HN q \
/ HOO I /
OCHy A further embodiment of a compound of the present invention is when R, is a chloro group, R2 is an OH group, R3 is methyl, RS and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-4-methoxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is methyl and R,o is isobutyl. The structure of this compound, Cryptophycin 8, is as follows:
- CI
i O
OH N
O / p f ~ CI
"O O
OCHa A further embodiment of a compound of the present invention is when Rl is a methoxy group, R2 is an OH group, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-4-methoxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is methyl and RIO is isobutyl. The structure of this compound, Cryptophycin 9, is as follows:

O
OH HN
O O CI

Ha A further embodiment of a compound of the present invention is when R, is a methoxy group, R2 and R4 are OH groups, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-4-methoxybenzyl, and R$
is a (2-carboxypropyl)amino group. The structure of this compound, Cryptophycin 10, is as follows:

0~ O
I OH OH
HN Cl :C
~N O I

HO O

A further embodiment of a compound of the present invention is when R, and R4 are taken together to form a tetrahydrofuran ring, R2 is an OH group, R3 is methyl, R5 and R6 are taken together to form a double bond between CS and C6. R7 is 3-chloro-4-methoxybenzyl, and R8 is a (2-carboxypropyl)amino group. The structure of this compound, Cryptophycin 12, is as follows:

j.jL i O
HN

H
HO O

A further embodiment of a compound of the present invention is when Rl and R, are taken together to form a double bond between the Clo and Cõ carbons, R3 is methyl, R4 is an OH group, P. and R6 are taken together to form a double bond between C5 and .
C6, R7 is 3-chloro-4-methoxybenzyl, and R8 is a (2-carboxypropyl)amino group.
The structure of this compound, Cryptophycin 14, is as follows:

\ \ / O

HN ~CI
~O N
~H ~~
HO' ~O

A further embodiment of a compound of the present invention is when R, and R2 are taken together to form an epoxide group, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R, is 3-chloro-4-hydroxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is methyl and Rlo is isobutyl. The structure of this compound, Cryptophycin 16, is as follows:

QLrO
O HN CI
N~c O OH

Y

A further embodiment of a compound of the present invention is when R, and R2 are taken together to form a double bond between C,o and Cõ carbons, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-4-hydroxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is methyl and R,o is isobutyl. The structure of this compound, Cryptophycin 17, is as follows:

O

co 00 HN a CI
~O
N~~ OH
H

A further embodiment of a compound of the present invention is when R, and R2 are taken together to form a double bond between C,o and Cõ carbons, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-4-methoxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is methyl and R,o is sec-butyl. The structure of this compound, Cryptophycin 18, is as follows:

o ~ % o a JOHN)( O
~H OCHa A further embodiment of a compound of the present invention is when R, and R, are taken together to form a double bond between C,o and Cli carbons, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-4-methoxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is methyl and R,o is isopropyl. The structure of this compound, Cryptophycin 19, is as follows:

I/ O 0 HN~ CI
NO , OCHa A further embodiment of a compound of the present invention is when R, and R2 are taken together to form an epoxide group, R3 is methyl, P. and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-4-methoxybenzyl, and R4 and R.
are taken together to form the didepsipeptide with the structure X where R9 is hydrogen and Rlo is isobutyl. The structure of this compound, Cryptophycin 21, is as follows:
QLO

o ~
~
oNo H OCN' ~

A further embodiment of a compound of the present invention is when R, and R2 are taken together to form an epoxide group, R3 is methyl, R5 and R6 are taken together to form a double bond between C. and C6, R7 is 3,5-dichloro-4-hydroxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X
where R9 is methyl and R,o is isobutyl. The structure of this compound, Cryptophycin 23, is as follows:

~ 0 O N~O
OH
~C' CI

A further embodiment of a compound of the present invention is when R, and R2 are taken together to form an epoxide group, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 4-methoxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is hydrogen and R,o is isobutyl. The structure of this compound, Cryptophycin 24, is as follows:

o I o ~
:~Oa ~Jv \N
H OCHa A further embodiment of a compound of the present invention is when R, and R2 are taken together to form a double bond between Clo and C11 carbons, R3 is methyl, R4 is hydroxy, R6 is hydrogen, R7 is 3-chloro-4-methoxybenzyl, and R. and R8 are taken together to form the didepsipeptide with the structure X where R9 is methyl and Rlo is isobutyl. The structure of this compound, Cryptophycin 26, is as follows:
O
HO O HN
~ CI
I O HN~O 00143 O"/ Y

CIiYPT (OPHYCIN-26 A further embodiment of a compound of the present invention is when Rl and R2 are taken together to form a double bond between the Clo and Cll carbons, R3 is hydrogen, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-4-methoxybenzyl, and R4 and R. are taken together to form the didepsipeptide with the structure X where R9 is methyl and Rlo is isobutyl. The structure of this compound, Cryptophycin 28, is as follows:

O OO HN
O 25 ~H

A further embodiment of a compound of the present invention is when RI and R2 are taken together to form a double bond between the Cio and Cl1 carbons, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-4-methoxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is hydrogen and Rlo is isobutyl. The structure of this compound, Cryptophycin 29, is as follows:

O
YO HN~p ~ G
' O v\N
H 0CHs CRYPTOPHYCiN-29 A further embodiment of a compound of the present invention is when R, and R2 are taken together to form a double bond between the Clo and Cll carbons, R3 is methyl, R5 is hydroxy, R6 is hydrogen, R7 is 3-chloro-4-methoxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is methyl and Rlo is isobutyl. The structure of this compound, Cryptophycin 30, is as follows:

o ~ O OH HN
p + ~ CI
O NO OCHa .

A further embodiment of a compound of the present invention is when R, and R, are taken together to form an epoxide group, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 3,5-dichloro-4-methoxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X
where R9 is methyl and R,o is isobutyl. The structure of this compound, Cryptophycin 31, is as follows:

O
vH CI
O ~'-~O CI

A further embodiment of a compound of the present invention is when Rl and R2 are taken together to form an epoxide group, R3 is methyl, R5 is hydrogen, R6 is hydrogen, R7 is 3-chloro-4-methoxybenzyl, and R4 and R$ are taken together to form the didepsipeptide with the structure X where R9 is methyl and R,o is isobutyl.
The structure of this compound, Cryptophycin 35, is as follows:
~ o I -p CI
O N~
~H OCHs A further embodiment of a compound of the present invention is when Rl and R2 are taken together to form an epoxide group, R3 is hydrogen, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-methoxybenzyl, and R4 and R.
are taken together to form the didepsipeptide with the structure X where R9 is methyl and R,o is isobutyl. The structure of this compound, Cryptophycin 40, is as follows:

o N
CI
'OV N~OOC~
H

CRYFrTOPHYCIN-40 A further embodiment of a compound of the present invention is when Rl and R.
are taken together to form a double bond between the Clo and C11 carbons, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 3,5-dichloro-4-hydroxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is methyl and Rlo is isobutyl. The structure of this compound, Cryptophycin 45, is as follows:

-01 C1r0 p HN
(,~
O''~0 H

A further embodiment of a compound of the present invention is when R, and R2 are taken together to form an epoxide group, R3 is methyl, R. and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-4-methoxybenzyl, and R4 and R.
are taken together to form the didepsipeptide with the structure X where R9 is methyl and R,o is propyl. The structure of this compound, Cryptophycin 49, is as follows:

O

Q HN ~,y N' :~O
OCHg A further embodiment of a compound of the present invention is when Rl and R2 are taken together to form a double bond between the CIo and C11 carbons, R3 is methyl, RS and R. are taken together to form a double bond between C. and C6, R7 is 3-chloro-4-methoxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is methyl and Rio is propyl. The structure of this compound, Cryptophycin 50, is as follows:

c o C 0 0 HN ~
a p N' C f /

A further embodiment of a compound of the present invention is when R, and R2 are taken together to form an epoxide group, R3 is methyl, R5 and R6 are taken together to form a double bond between C5 and C6, R7 is 3-chloro-4-methoxybenzyl, and R4 and R8 are taken together to form the didepsipeptide with the structure X where R9 is methyl and Rlo is sec-butyl. The structure of this compound, Cryptophycin 54, is as follows:

o I ~ 0 8 ~
N~
OCHs Of the above compounds, Cryptophycins 2, 4, 16-19, 21, 23, 24, 26, 28-31, 40, 43, 45, 49, 50, and 54 are metabolites produced by a strain of Nostoc sp. of blue-green algae (cyanobacteria) which has been cultured, with these compounds subsequently isolated from this culture. Cryptophycins 6 and 7 are artifacts that are produced if the isolation procedure utilizes solvents containing methanol. Cryptophycins 8, 9, 10-12, 14, and 35 are derivatives of these naturally-produced metabolites, having been chemically modified with the methods described in the Experimental Section of this application, with alternate methods to create the exemplified compounds, as well as the non-exemplified compounds, available to those of ordinary skill in the art.
The present invention provides methods of producing the above cryptophycin compounds through the culturing of a strain of the Nostoc sp. The morphological characteristics of the Nostoc sp. of blue-green algae (cyanobacteria), as provided in U.S.
Patent No. 4,946,835, are that they are filamentous and consist of vegetative cells. In longer filaments, heterocysts occasionally are observed in an intercalary position; akinetes are not observed. Reproduction is by hormogonia in addition to random trichome breakage. The basis for an identification of a Nostoc sp. can be found in J.
Gen. Micro., 111:1-61 (1979).

The invention further provides that a Nostoc sp. may be cultured and that novel cryptophycin metabolites, as well as previously disclosed cryptophycin metabolites, may be isolated from this culture. In a preferred embodiment of the present invention, the Nostoc sp. strain designated GSV 224 is the strain which is cultivated and from which are isolated compounds represented by the following structure:
Ri F~ R6 C
Fia O
Wherein Rl is H, OH, a halogen, 0 of a ketone group, NH2, SH, a lower alkoxyl group or a lower alkyl group;
R2 is H, OH, 0 of a ketone group, NH2, SH, a lower alkoxyl group or a lower alkyl group; or R, and R2 may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring or a double bond between Cio and C11; or Rl and R4 may be taken together to form a tetrahydrofuran ring;
R3 is H or a lower alkyl group;
R4 is OH, a lower alkanoyloxy group or a lower a-hydroxy alkanoyloxy group;
R5 is H or an OH group;
R6 is H; or R5 and R6 may be taken together to form a double bond between C5 and C6;
R7 is a benzyl, hydroxybenzyl, methoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, halomethoxybenzyl, or dihalomethoxybenzyl group;
R. is OH, a lower j6-amino acid wherein Cl is bonded to N of the (3-amino acid, or an esterified lower 0-amino acid wherein C, is bonded to N of the esterified lower 0-amino acid group;

R4 and R8 may be taken together to form a didepsipeptide group consisting of a lower /3-amino acid bonded to a lower a-hydroxy alkanoic acid; or R5 and R. may be taken together to form a didepsipeptide group consisting of a lower /3-amino acid bonded to a lower a-hydroxy alkanoic acid; 5 with the following provisos:

Rl is H, a lower alkyl group, or a lower alkoxyl group only if R2 is OH, 0 of a ketone group, NH2, SH.
In a preferred embodiment of the invention, chemically modifying a cryptophycin metabolite isolated by the above method provides a distinct compound also having this structure. Procedures for chemically modifying cryptophycin compounds to produce additional compounds within the scope of the present invention are available to those of ordinary skill in the art. Moreover, additional procedures are described in greater detail in the Experimental Section of this application.
In addition to the novel cryptophycin compounds of the present invention, the present invention provides novel methods of producing, as well as using, the above structure which includes the following previously disclosed cryptophycin species, Cryptophycins 1, 3, 5, 13 and 15. The structures of these compounds are as follows:
H
O
O =H =
O~ O O HN CI
O N~O I /

~ O

O O O HN CI
= p N~p + /
H

H
C O
O :
OH O HN q OH N~p /
\~~
CHsO CH
O
CRYPTOPHYCIN S
H

CI
H Np OCH~
H
HO O

OH
O
- I / OH d H
O O C~

The invention provided herewith is directed to any strain of the Nostoc sp.
and preferably to the Nostoc sp. GSV 224 strain to produce cryptophycin compounds.
To that end, the GSV 224 strain of Nostoc sp. was deposited on October 7, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC
Accession No. 55483. Other strains of Nostoc sp., in particular strain MB 5357 previously deposited by Merck and Co. under ATCC accession No. 53789, are strains contemplated to be utilized to practice the present invention.
As is the case with other organisms, the characteristics of Nostoc sp. are subject to variation. For example, recombinants, variants, or mutants of the specified strains may be obtained by treatment with various known physical and chemical mutagens, such as ultraviolet ray, X-rays, gamma rays, and N-methyl-N'-nitro-N-nitrosoguanidine.
All natural and induced variants, mutants, and recombinants of the specified strains which retain the characteristic of producing a cryptophycin compound are intended to be within the scope of the claimed invention.
The cryptophycin compounds of the present invention can be prepared by culturing a strain of Nostoc sp. under submerged aerobic conditions in a suitable culture medium until substantial antibiotic activity is produced. Other culture techniques, such as surface growth on solidified media, can also be used to produce these compounds. The culture medium used to grow the specified strains can include any of one of many nitrogen and carbon sources and inorganic salts that are known to those of ordinary skill in the art.
Economy in production, optimal yields, and ease of product isolation are factors to consider when choosing the carbon sources and nitrogen sources to be used.
Among the nutrient inorganic salts which can be incorporated in the culture media are the customary soluble salts capable of yielding iron, potassium, sodium, magnesium, calcium, ammonium, chloride, carbonate, phosphate, sulfate, nitrate, and like ions.
Essential trace elements which are necessary for the growth and development of the organisms should also be included in the culture medium. Such trace elements commonly occur as impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of the organisms. It may be desirable to add small amounts (i.e. 0.2mL/L) of an antifoam agent such as polypropylene glycol (M.W.
about 2000) to large scale cultivation media if foaming becomes a problem.
For production of substantial quantities of the cryptophycin compounds, submerged aerobic cultivation in tanks can be used. Small quantities may be obtained by shake-flask culture. Because of the time lag in metabolite production commonly associated with inoculation of large tanks with the organisms, it is preferable to use a vegetative inoculum. The vegetative inoculum is prepared by inoculating a small volume of culture medium with fragments of the vegetative trichome or heterocyst-containing form of the organism to obtain a fresh, actively growing culture of the organism. The vegetative inoculum is then transferred to a larger tank. The medium used for the vegetative inoculum can be the same as that used for larger cultivations or fermentation, but other media can also be used.
The organisms may be grown at temperatures between about 20' C and 30' C and an incident illumination intensity of about 100 to 200 mol photons ni 2Sec-1 (photosynthetically active radiation).
As is customary in aerobic submerged culture processes of this type, carbon dioxide gas is introduced into the culture by addition to the sterile air stream bubbled through the culture medium. For efficient production of the cryptophycin compounds, the proportion of carbon dioxide should be about 1%(at 24' C and one atmosphere of pressure).
The prior art, specifically U.S. Patent No. 4,946,835, provides methods of cultivating Nostoc sp., the contents of which are hereby incorporated by reference.
Cryptophycin compound production can be followed during *the cultivation by testing samples of the broth against organisms known to be sensitive to these antibiotics.
One useful assay organism is Candida albicans.
Following their production under submerged aerobic culture conditions, cryptophycin compounds of the invention can be recovered from the culture and from the culture media by methods known to those of ordinary skill in this art.
Recovery is generally accomplished by initially filtering the culture medium to separate the algal cells and then freeze-drying the separated cells. The freeze-dried alga can be extracted with a - suitable solvent such as ethanol, methanol, isopropanol, or dichloromethane.
The cryptophycins can be separated by subjecting this extract, as well as the culture media, to rapid chromatography on reversed-phase column. The cryptophycins can be purified by reversed-phase high-performance liquid chromatography (HPLC).
As will be apparent from their structures, the cryptophycin compounds have groups which are capable of chemical modification. The genus compound of the present invention contemplates those cryptophycins which exhibit anti-neoplastic activity. For example, the derivatives exemplified in the present invention include compounds having the epoxide oxygen or hydroxy groups on C-7 and C-8 of unit A or the leucic acid group of unit B of Figure 1. Such derivatives of the novel and previously disclosed compounds which display the desired anti-neoplastic activity are included in the claimed invention.
Moreover, the relationship between the structure of the cryptophycin compounds and anti-neoplastic activity is provided in the Experimental Section hereinbelow.
While selected cryptophycin compounds are known to be metabolites produced by the alga of the present invention, other cryptophycin compounds, e.g.
Cryptophycins 8-15, can be derived from the metabolites using published techniques which are known to those of ordinary skill in the art; for example, the syntheses disclosed in U.S. Patent Nos.
4,868,208, 4,845,086, and 4,845,085, the contents of which are hereby incorporated by reference, or by utilizing other methods which are known to those of ordinary skill in the art. Moreover, the present invention provides methods of producing derivatives in the Experimental Section.
Cryptophycins are potent antitumor and antifungal depsipeptides from blue-green algae (cyanobacteria) belonging to the Nostocaceae. The first cryptophycin, Cryptophycin 1, was isolated from terrestrial Nostoc sp. ATCC 53789 and found to be very active against fungi, especially strains of Cryptococcus (R.E. Schwartz et al., J.
Ind. Microbiol.
1990, 5:113-124). Cryptophycin 1 has also been isolated from terrestrial Nostoc sp. GSV
224, along with twenty-four additional cryptophycin analogs as minor constituents of the alga, and found to be very active against subcutaneously transplanted solid tumors in mice (G. Trimurtulu et al., J. Am. Chem. Soc. 1994, 116:4729-4737; R. Barrow et al., J. Am.
Chem. Soc. 1995, 117:2479-2490). Two of the analogs from Nostoc sp. GSV 224, Cryptophycins 3 and 5, had been described previously as semi-synthetic analogs of Cryptophycin 1 (D.F. Sesin, U.S. Patent 4,845,085, issued July 4, 1989; D.F.
Sesin et al., U.S. Patent 4,868,208, issued September 19, 1989). The cryptophycins showed significant tumor selective cytotoxicity in the Corbett assay and were equally cytotoxic against drug 'sensitive and drug resistant tumor cells. Cryptophycin 1 appeared to have the same mode of action as vinblastine, but differed from the latter drug in irreversibly inhibiting microtubule assembly (C:D. =Smith et ai., Cancer Res. 1994, 54:3779-3784).
One of the cryptophycins from Nostoc sp. GSV 224, Cryptophycin 24, has been isolated from a marine sponge and named arenastatin A (M. Kobayashi et al:, Tetrahedrori l.ett.
1994, 35:7969-72; M. Kobayashi et al., Tennen Yuki Kagobutsu Toronkai Koen Yoshishu 1994, .36st, 104-110).
Twenry-two additional cryptophycin compounds, herein designated 'Cryptophycins 2, 4, b, 7, 16-19, 21, 23; 24, 26, 28-31, 40, 43, 45, 49, 50 and 54 are disclosed in International Patent Application VKO/1995/017093 ftled December 21, 1994, such compounds eitheF being metabolites isolated from a strain of Nostoc sp; or having been semi-synthesized from such-metabolites. Also disclosed in these patent applications is the -characteriiation of selected cryptophycin compounds' as anti-microtubule agents with clinical-type activity expressed toward a broad spectrum of tumors implanted in mice, including DR and MDR tumors.
The prescnt invention provides novel cryptophycin compounds having the following structure: -.
Ri R2 R9 q5 qr O

X O HN Ra ' Ro 3CY 'R, HN O

O
P-i Re wherein Ar is methyl or phenyl or any simple unsubstituted or substituted aromatic or heteroaromatic group;
Rl is a halogen, SH, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, or phosphate;
R2 is OH or SH; or Rl and R2 may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring or a monoalkylphosphate ring; or Rl and R2 may be taken together to form a double bond between Cl$ and C19;
R3 is a lower alkyl group;
R4andR5areH;or R4 and R5 may be taken together to form a double bond between C13 and C14;
R6 is a benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, haloalkoxybenzyl, or dihaloalkoxybenzyl group;
R7, R8, R9 and Rlo are each independently H or a lower alkyl group; and X and Y are each independently 0, NH or alkylamino.
In a preferred embodiment of this cryptophycin, R8 of the cryptophycin is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl. In another preferred embodiment of this cryptophycin, R7 is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl.
In an additional preferred embod'unent of this cryptophycin, R7 is H, R. is methyl, R3 is methyl; X and Y are not both O.
The present invention provides an additional preferred embodiment of this cryptophycin wherein R3 is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl.
In an another preferred embodiment of this cryptophycin, R9 is methyl, ethyl, propyl, butyl, isobutyl, pentyl or isopentyl. In a further preferred embod'unent of this cryptophycin, Rlo is methyl, ethyl, propyl, butyl, isobutyl, pentyl or isopentyl.
The invention further provides cryptophycin compounds wherein at least one of the groups attached to C3, C6, C,o, C16, C,7, and Cl8 has R stereochemistry. In a further embodiment of the invention, at least one of the groups attached to C3, C61 C,o, C162 C17, and C18 has S stereochemistry.

One embodiment of a cryptophycin compound of the present invention is when Ar is phenyl, R, and R2 are taken together to form a double bond between C18 and C19, R3, R7 and R8 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, Rg is isobutyl, Rlo is hydrogen, and X and Y
are oxygen. The structure of this compound, Cryptophycin 51, is as follows:

O
O
O HN ==~'' ci O N O
~-----H OCH3 A further embodiment of a compound of the present invention is when Ar is phenyl, Ri and R2 are taken together to form an R,R-epoxide ring, R3, R7 and R8 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, Rlo is hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 52, is as follows:
O
. I / O O
ci O HN C
O N

H

A further embodiment of a compound of the present invention is when Ar is phenyl, Rl and R2 are taken together to form an S, S-epoxide ring, R3, R7 and R8 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, RIo is hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 53, is as follows:
- - ,Q

I / O
O HN ci I0NO ~
= H OCH3 A further embodiment of a compound of the present invention is when Ar is phenyl, R, is S-chloro, R2 is R-hydroxyl, R3, R7 and R8 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R,o is hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 55, is as follows:

CI

I/ O OH O
O HN CI
O N
= H OCH3 Another embodiment of a compound of the present invention is when Ar is phenyl, R, and R2 are taken together to form an R,R-epoxide ring, R3, R7 and R8 are methyl, R4 and R5 are hydrogen, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R10 is hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 57, is as follows:

I / O O
O HN ci O N

Another embodiment of a compound of the present invention is when Ar is phenyl, Rl is S-chloro, R2 is R-hydroxyl, R3, R7 and R. are methyl, R4 and R5 are hydrogen, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R,o is hydrogen, and X and Y
are oxygen. The structure of this compound, Cryptophycin 58, is as follows:
CI
O
OOH O
O HN ci I0c O H OCH3 Another embodiment of a compound of the present invention is when Ar is phenyl, Rl and R. are taken together to form an R, R-episulfide ring, R3, R7 and Ra are methyl, R,, and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R,o is hydrogen, and X and Y are oxygen. The structnre of this compound, Cryptophycin 61, is as follows:
S
101~ O

O O HN CI
O HN : O aOMe O

Another embodiment of a compound of the present invention is when Ar is p-methoxyphenyl, Rl and R2 are taken together to form a double bond between Cls and C19, R3 and R7 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, Rs is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R$ and Rlo are hydrogen, and X
and Y are oxygen. The structare of this compound, Cryptophycin 81, is as follows:
O
O O HN CN
Me0 O HN O ~ OMe Another embodiment of a compound of the present invention is when Ar is methyl, Ri and R2 are taken together to form a double bond between Cla and C19, R3 and R7 are methyl, R4 and RS are taken together to form a double bond between C13 and C14, Rs is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R8 and R,o are hydrogen, and X
and Y
are oxygen. The structure of this compound, Cryptophycin 82, is as follows:
O
O HN

O HN O OMe O

Another embodiment of a compound of the present invention is when Ar is methyl, Rl and R2 are taken together to form an R,R-epoxide ring, R3 and R7 are methyl, R4 and RS are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R$ and Rlo are hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 90, is as follows:
O
O O HO O

Another embodiment of a compound of the present invention is when Ar is methyl, Rl and R2 are taken together to form an S, S-epoxide ring, R3 and R7 are methyl, R4 and RS are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R8 and RIO are hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 91, is as follows:

O
cx: :xtc:
O O

Another embodiment of a compound of the present invention is when Ar is phenyl, Ri and R2 are taken together to form an R,R-aziridine ring, R3, R7 and R8 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, Rg is isobutyl, Rlo is hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 97, is as follows:

NH

O O HN CI
.==~~\\
O HN O ~ OMe Another embodiment of a compound of the present invention is when Ar is p-fluorophenyl, R, and R2 are taken together to form a double bond between Cls and C29, R3 and R7 are methyl, R4 and Rs are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R8 and Rlo are hydrogen, and X
and Y are oxygen. The structure of this compound, Cryptophycin 110, is as follows:
O
p O :Xo:
W
O

Another embodiment of a compound of the present invention is when Ar is p-tolyl, Rl and R2 are taken together to form a double bond between C,$ and C19, R3 and R7 are methyl, R4 and R5 are taken together to form a double bond between C,3 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R$ and Rio are hydrogen, and X and Y
are oxygen. The structure of this compound, Cryptophycin 111, is as follows:
O
O O HN
. NIa O HN O aOw O

Another embodiment of a compound of the present invention is when Ar is 2-thienyl, Rl and R2 are taken together to form a double bond between Cla and C19, R3 and R7 are methyl, R4 and Rs are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, Rs and Rlo are hydrogen, and X
and Y
are oxygen. The structure of this compound, Cryptophycin 112, is as follows:
S O
p O HN

O HN O Ow O

Another embodiment of a compound of the present invention is when Ar is p-fluorophenyl, Rl and R2 are taken together to form an R, R-epoxide ring, R3 and R7 are methyl, R4 and RS are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R8 and Rlo are hydrogen, and X and Y
are oxygen. The structure of this compound, Cryptophycin 115, is as follows:
O O
O HN \ a O

Another embodiment of a compound 'of the present invention is when Ar is p-fluorophenyl, Ri and R2 are taken together to form an S, S-epoxide ring, R3 and R7 are methyl, R4 and Rs are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R$ and R,o are hydrogen, and X and Y
are oxygen. The structure of this compound, Cryptophycin 116, is as follows:
O
O O HN

,O HN O

Another embodiment of a compound of the present invention is when Ar is p-tolyl, Rl and R2 are taken together to form an R,R-epoxide ring, R3 and R7 are methyl, R4 and RS are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, Ra and R,a are hydrogen, and X and Y are oxygen. The sMature of this compound, Cryptophycin 117, is as follows:

O O HN ,, aCl Ms .
O HN O OMe _42_ Another embodiment of a compound of the present invention is when Ar is p-tolyl, Rl and R2 are taken together to form an S, S-epoxide ring, R3 and R7 are methyl, R4 and RS are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R8 and R,o are hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 118, is as follows:
.14 O
~
Lo HN CI
Me O HN O LL/ OMe O

Another embodiment of a compound of the present invention is when Ar is 2-thienyl, Rl and R2 are taken together to form an R,R-epoxide ring, R3 and R7 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R8 and R,o are hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 119, is as follows:
O

~ I O O HN õ~~~~ CI
I
)%0 HN O / OMe O

Another embodiment of a compound of the present invention is when Ar is 2-thienyl, Rl and R2 are taken together to form an S, S-epoxide ring, R3 and R7 are methyl, R; and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, Rg is isobutyl, R8 and R,o are hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 120, is as follows:

S

~ I C p HN
I /
p HN O OMe O

Another embodiment of a compound of the present invention is when Ar is phenyl, Rl and R2 are taken together to form a double bond between C18 and C19, R3 and R7 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R$ and Rlo are hydrogen, X is oxygen and Y is a nitrogen bearing a single hydrogen. The structure of this compound, Cryptophycin 121, is as follows:

O
O O HN ~
' ~~~

NH HN O / O~
O

Another embodiment of a compound of the present invention is when Ar is phenyl, R, and R2 are taken together to form an R,R-epoxide ring, R3 and R7 are methyl, R4 and RS are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R8 and Rlo are hydrogen, X is oxygen and Y is a nitrogen bearing a single hydrogen. The structure of this compound, Cryptophycin 122, is as follows:
O
O
O O HN C O
NH HN / OMe O

Another embodiment of a compound of the present invention is when Ar is phenyl, Rl and R2 are taken together to form an S, S-epoxide ring, R3 and R7 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R8 and R,o are hydrogen, X is oxygen and Y is a nitrogen bearing a single hydrogen. The structure of this compound, Cryptophycin 123, is as follows:

.111\2 O
O O HN Cl ..J NH HN :: O / OMe O

Another embodiment of a compound of the present invention is when Ar is p-chlorophenyl, R, and R2 are taken together to form a double bond between Cls and C19, R3 and R7 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14i R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, Rs and Rio are hydrogen, and X
and Y are oxygen. The structure of this compound, Crvptonhvcin 124. is ac fntlows:
O
O O HN a a (il O HN O
O

Another embodiment of a compound of the present invention is when Ar is p-chlorophenyl, Rl and R2 are taken together to form an R,R-epoxide ring, R3 and R7 are methyl, R4 and RS are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, Rs and Rlo are hydrogen, and X and Y
are oxygen. The structure of this compound, Cryptophycin 125, is as follows:
O O
.=='~~~
p O HN a a O HN O ~ OMe Another embodiment of a compound of the present invention is when Ar is p-chiorophenyl, R, and R2 are taken together to form an S S epoxide ring, R3 and R7 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R8 and Rlo are hydrogen, and X and Y
are oxygen. The structure of this compound, Cryptophycin 126, is as follows:
.142 O
O O HN .~~~~~ CI
G't O HN O / OMe CRYPTOPIiYCIN 126 Another embodiment of a compound of the present invention is when Ar is phenyl, RI is S-chloro, R2 is R-hydroxyl, R3 and R7 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R$ and RIO are hydrogen, X is oxygen and Y is a nitrogen bearing a single hydrogen. The structure of this compound, Cryptophycin 127, is as follows:
O
OH O O HN ::M 25 NH HN OMe O

Another embodiment of a compound of the present invention is when Ar is p-tolyl, Rl is S-chloro, R2 is R-hydroxyl, R3 and R7 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, Ra and R,o are hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 128, is as follows:
Cf O
q.{ p O :xc:
N 10 O Me Another embodiment of a compound of the present invention is when Ar is p-tolyl, Rl is R-chloro, R2 is S-hydroxyl, R3 and R7 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R$ and Rlo are hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 130, is as follows:
cl a H 0 O HN CI
.=
NIa O HN O OMe O

Another embod'unent of a compound of the present invention is when Ar is p-tolyl, Rl is R-chloro, R2 is R-hydroxyl, R3 and R7 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, Ra and R,o are hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 131, is as follows:
C:I
O
O!-1 O O HN a O HN O O~
O

Another embodiment of a compound of the present invention is when Ar is p-chlorophenyl, Rl is S-chloro, R2 is R-hydroxyl, R3 and R7 are methyl, R4 and RS are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, Rg is isobutyl, R8 and Rio are hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 132, is as follows:
q O
OH O O HN

O HN I: O ~ OMe O f-ly Another embodiment of a compound of the present invention is when Ar is p-chlorophenyl, Rl is R-chloro, R2 is S-hydroxyl, R3 and R7 are methyl, R4 and Rs are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, Ra and Rlo are hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 133, is as follows:

O
OH O O HN a CI
O HN O Ow Another embodiment of a compound of the present invention is when Ar is p-chlorophenyl, RI is R-chloro. R2 is R-hydroxyl, R3 and R7 are methyl, R4 and R5 are taken together to form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R8 and R,o are hydrogen, and X and Y are oxygen. The structure of this compound, Cryptophycin 134, is as follows:
O
CH O tzc 0 0 OMe O

Set forth hereinbelow are additional cryptophycin compounds, their substituent groups based upon the following structure:

R,~ ae <
O~FR12 o ~-O R* HN ~
~
~~H o + ~ ~Fb Wherein Rl is H or a halogen;
R2 is H, an oxygen of a ketone or OH; or R, and R2 may be taken together to form an epoxide ring; or R, and R2 may be taken together to form an episulfide ring;
R3 is H, or a lower alkyl group;
R4isHorOH;
R5 is H or OH; or R4 and R5 may be taken together to form a double bond;
R6 is H or a halogen;
With the following proviso when R, and R2 are taken together to form an epoxide group, R4 and R5 are taken together to form a double bond and R6 is chlorine, R3 is not methyl.

An embodiment of a cryptophycin compound of the present invention is when Rl is hydrogen, R2 is an oxygen of a ketone group, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 20 is as follows:

o I
CI
O
HNO
OCHs ~J
O

A further embodiment of a compound of the present invention is when R, is S-bromo, R2 is R-hydroxy, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 25 is as follows:
Br o 7,~O O HN~ a HN O-------\OCH3 OIY

A further embodiment of a compound of the present invention is when Rl is R-chloro, R2 is R-hydroxy, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 27 is as follows:
ci i -OHrOHN
O HN~ G
o ~J
CR1fPTOPHYCIN 27 A further embodiment of a compound of the present invention is when R, and R2 are taken together to form a R, R-epoxide ring, R3 is S-methyl, R4 and R5 are hydrogen and R6 is chloro. The structure of this compound, Cryptophycin 32 is as follows:

O
_ o HN G
O HN)~ I
O~~ ~~

A further embodiment of a compound of the present invention is when R,, R4 and R5 are hydrogen, R2 is S-hydroxy, R3 is R-methyl and R6 is chloro. The structure of this compound, Cryptophycin 33 is as follows:

\ o I
CI
/ ~~n O HN
HN)~O
C"3 OJY

A further embodiment of a compound of the present invention is when Rl, R,, R4 and R5 are hydrogen, R3 is R-methyl and R6 is hydrogen. The structure of this compound, Cryptophycin 34 is as follows:

o _ CHr ~
O HN O \% , A further embodiment of a compound of the present invention is when R, is R-bromo, R2 is R-hydroxy, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 37 is as follows:
Br O OH O HN CI

O HN~O \ /
H

A further embodiment of a compound of the present invention is when RI and R2 are taken together to form a S, S-epoxide ring, R3 is S-methyl, R4 and RS are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 38, is as follows:

O
I _ O HN
~'~~ ~.. ! CI
O HN
OCH~
O
~J

A further embodiment of a compound of the present invention is when R, and R2 are taken together to form a S,R-epoxide ring, R3 is S-methyl, R4 and R. are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 39, is as follows:

o O HN C~

O HN~O yi ~ J C~
0i +Y

A further embodiment of a compound of the present invention is when RI and R2 are taken together to form a R, R-epoxide ring, R3 is S-methyl, R4 is S-hydroxy, R5 is R-hydroxy and R6 is chloro. The structure of this compound, Cryptophycin 41, is as follows:

OH
a OH HN CI
O y O HNO /
O~
~~

A further embodiment of a compound of the present invention is when R, and R2 are taken together to form a R,R-epoxide ring, R3 is S-methyl, R4 is R-hydroxy, R5 is S-hydroxy and R6 is chloro. The structure of this compound, Cryptophycin 42, is as follows: 5 OH
O
H FiN ci O ~
~
O HNp / OCj3 O-1y A further embodiment of a compound of the present invention is when R, is hydrogen, R2 is S-hydroxy, R3 is R-methyl, R4 and RS are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 48, is as follows:

OHN
CI
HN O
~ J ~~
O

A further embodiment of a compound of the present invention is when R, is S-chloro, R2 is R-hydroxy, R3 is S-methyl, R4 and R5 are hydrogen and R6 is chloro. The structure of this compound, Cryptophycin 59, is as follows:

C'OH
. \ O
-O HN ~
O HN
Op I i ~J CH3 A further embodiment of a compound of the present invention is when Rl and R2 are taken together to form a S, S-episulfide ring, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 60, is as follows:

o O HN ci .'~
O HN:CO

A further embodiment of a compound of the present invention is when Rl is S-chloro, R2 is R-hydroxy, R3 is hydrogen, R4 and R5 are taken together to form a double bond and Rs is chloro. The structure of this compound, Cryptophycin 63, is as follows:
~ OH
O
O HN
a O HNO

A further embodiment of a compound of the present invention is when Rl is R-chloro, R2 is R-hydroxy, R3 is S-methyl, R4 and R5 are hydrogen and R6 is chloro. The structure of this compound, Cryptophycin 64, is as follows:

CI
OH

O HN
"~O HN)~ Tl\
O~ O ~ a Hs A further embodiment of a compound of the present invention is when R, is R-chloro, R2 is S-hydroxy, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 69, is as follows:
= c_i o Cr OH O HN a O HN~CO
C"3 A further embodiment of a compound of the present invention is when R, is S-chloro, R2 is S-hydroxy, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 70, is as follows:
CI
i O
CHOHN
CI

L"~O HN~O
oclis O-Iy CRYPTaPHYCIN 70 A further embodiment of a compound of the present invention is when R, is R-bromo, R2 is S-hydroxy, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 71, is as follows:
Br O
, _ -OH O HN
~ CI
O HN~O

O
-Y

A further embodiment of a compound of the present invention is when Rl is S-bromo, R2 is S-hydroxy, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 72, is as follows:
cJZ1rOHN0 ~.. cl O HN O \~..

A further embodiment of a compound of the present invention is when RI is S-chloro, R2 is S-hydroxy, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 73, is as follows:
OH
O
HN

O HNO

O--J~

A further embodiment of a compound of the present invention is when R, is S-chloro, R2 is R-hydroxy, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is hydrogen. The structure of this compound, Cryptophycin 74, is as follows:

ci = OH
O
O HN

O HN' O

O~y A further embodiment of a compound of the present invention is when R, is S-fluoro, R2 is R-hydroxy, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 75, is as follows:
F
O

7t'~~ HN ~
HNO

CRl(PTOPHYCIN 75 A further embodiment of a compound of the present invention is when R, is R-fluoro, R2 is R-hydroxy, R3 is S-methyl, R4 and R5 are taken together to form a double bond and R6 is chloro. The structure of this compound, Cryptophycin 76, is as follows:
F
O
HN q IY
HN

J,J

The present invention provides methods of producing the above cryptophycin compounds, as well as all previously known cryptophycins, through total synthesis.
The invention further provides that novel cryptophycin metabolites, as well as previously disclosed cryptophycin metabolites, may be synthesized using the methods provided in this invention.
The present invention provides a method for producing a cryptophycin comprising selecting an allylically substituted E alkene; rearranging the allylically substituted E
alkene via a stereospecific Wittig rearrangement; converting this compound to a first 8-_ amino acid or 6-hydroxy acid; coupling the first acid to a second a-amino acid to form a first subunit; coupling a third j6-amino acid to a fourth a-hydroxy acid or a-amino acid to form a second subunit; and coupling the first subunit to the second subunit to form a cryptophycin.
The present invention further provides a preferred embodiment of the method, wherein the cryptophycin produced has the following structure:
RiR2R3 ~~
Ar 'g ' 13 O

X HN Rs Rio Y HN O
O
~-i R8 wherein Ar is methyl or phenyl or any simple unsubstituted or substituted aromatic or heteroaromatic group;
Rl is a halogen, SH, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, or phosphate;
R2 is OH or SH; or Rl and R2 may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring or a monoalkylphosphate ring; or Rl and R2 may be taken together to form a double bond between C18 and C19;
R3 is a lower alkyl group; 5 R4andR5areH;or R4 and R5 may be taken together to form a double bond between C13 and C14; R6 is a benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, haloalkoxybenzyl, or dihaloalkoxybenzyl group;
R7, R8, R9 and Rlo are each independently H or a lower alkyl group; and X and Y are each independently 0, NH or alkylamino.
In a preferred embodiment of the present invention, the method produces a cryptophycin wherein Ar is phenyl; R3 is methyl; R6 is halomethoxybenzyl; R7 is H; R. is methyl; R9 is isobutyl; R,o is H; X is 0; and Y is O.
In addition to the present invention providing cryptophycins with the above structure, the present invention provides methods of producing previously disclosed cryptophycins and prior art cryptophycins. Cryptophycins 1, 8 and 35 were produced by total synthesis. Provided hereinbelow is a representation of previously disclosed and prior art cryptophycins produced by total synthesis:

RI R2 ~ ~
O
):X: ::ccc:H3 OIY

wherein R, is a halogen; R2 is OH; or Rl and R2 may be taken together to form an epoxide ring;
R3 is H; and R4 is H; or R3 and R4 may be taken together to form a double bond.
The present invention also provides a pharmaceutical composition useful for inhibiting the proliferation of a hyperproliferative mammalian cell comprising an effective amount of a cryptophycin with the following structure:

Ri R2 R3 ~ R5 Ar 'a 13 O
,e ,q X O HN Rs ~ 3 Rio Y HN O
O e wherein Ar is methyl or phenyl or any simple unsubstituted or substituted aromatic or heteroaromatic group;
RI is a halogen, SH, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, or phosphate;
R2 is OH or SH; or Rl and R2 may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring or a monoalkylphosphate ring; or Rl and R2 may be taken together to form a double bond between C18 and C19;
R3 is a lower alkyl group;
R4andR5areH; or R4 and R5 may be taken together to form a double bond between C13 and C14;

R6 is a benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, haloalkoxybenzyl, or dihaloalkoxybenzyl group;
R7, R8, R9 and R,o are each independently H or a lower alkyl group; and X and Y are each independently 0, NH or alkylamino;
together with a pharmaceutically acceptable carrier.
In a preferred embodiment of the present invention, the pharmaceutical composition further comprises at least one additional anti-neoplastic agent.
The present invention also provides a method for inhibiting the proliferation of a mammalian cell comprising contacting the mammalian cell with a cryptophycin compound in an amount sufficient to inhibit the proliferation of the cell, the cryptophycin compound having the following structure:

RiR2R3 R5 Ar 19 ' 13 O

X O HN Rs ~ 3 3 Rio Y HN O

wherein Ar is methyl or phenyl or any simple unsubstituted or substituted aromatic or heteroaromatic group;
Rl is a halogen, SH, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, or phosphate;
R2 is OH or SH; or Rl and R2 may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring or a monoalkylphosphate ring; or Rl and R2 may be taken together to form a double bond between C18 and C19i R3 is a lower alkyl group;
R4 and R5 are H; or R4 and R5 may be taken together to form a double bond between C13 and C14;
R6 is a benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, haloalkoxybenzyl, or dihaloalkoxybenzyl group;
R7, R8, R9 and RIO are each independently H or a lower alkyl group; apd X and Y are each independently 0, NH or alkylamino.
In a preferred embodiment of the present invention, this method further comprises contacting the cell with at least one additional anti-neoplastic agent. In a preferred embodiment of the present invention, the mammalian cell contacted is hyperproliferative.
In a further preferred embodiment of the present invention, the hyperproliferative cell is human.

The present invention also provides a method of inhibiting the proliferation of a hyperproliferative mammalian cell having a multiple drug resistant phenotype comprising contacting the cell with an amount of a cryptophycin compound effective to disrupt the dynamic state of microtubule polymerization and depolymerization to arrest cell mitosis, thereby inhibiting the proliferation of the cell, the cryptophycin compound having the following structure:

RtR2R3 ~~
Ar 1 1 13 O
Ie ie 'a X HN Rs Rto Y HN O

wherein Ar is methyl or phenyl or any simple unsubstituted or substituted aromatic or heteroaromatic group;
Rl is a halogen, SH, amino, monoalkylamino, diallcylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, or phosphate;
R2 is OH or SH; or Rl and R2 may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring or a monoalkylphosphate ring; or Rl and R2 may be taken together to form a double bond between C18 and C19;
R3 is a lower alkyl group;
R4andR5are H; or R4 and R5 may be taken together to form a double bond between C13 and C14;
R6 is a benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, T
haloalkoxybenzyl, or dihaloalkoxybenzyl group;

R7, R8, R9 and Rlo are each independently H or a lower alkyl group; and X and Y are each independently 0, NH or alkylamino.
In a preferred embodiment of the present invention, the method further comprises contacting the cell with at least one additional anti-neoplastic agent. In a further preferred embodiment of the present invention, the mammalian cell is human.
The present invention also provides a method of alleviating a pathological condition caused by hyperproliferating mammalian cells comprising administering to a subject an effective amount of the pharmaceutical composition disclosed herein to inhibit proliferation of the cells. In a preferred embodiment of the present invention, the mammalian cells are human.
In preferred embodiment of the present 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 mammory, small-cell lung, non-small-cell lung, colorectal, leukemia, melanoma, pancreatic adenocarcinoma, 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.
The method for preparing the cryptophycin compounds is summarized in Scheme 1 as depicted in Figure 5. The starting material is a 3E-alkene (a) substituted at C-2 with a XH group in the S-configuration where X is oxygen or NH. L-Alanine and L-lactic acid serve as inexpensive sources of the starting material a. The key step in the synthesis is a stereoselective [2,3] Wittig rearrangement (D.J-S. Tsai et al., J. Org. Chem.
1984, 49:1842-1843; K. Mikami et al., Tetrahedron 1984, 25:2303-2308; N. Sayo et al., Chem Lett. 1984, 259-262) of the propargyl ether of a (b) to the (3R,4R)-3-(XH-substituted)-4-alkylhept-5(E)-en-1-yne (c) where X is an oxygen or a protected nitrogen (e.g.
t-butyldimethylsilylamino). Compound c can then be converted to the 8-hydroxy or amino acid unit A precursor of the cryptophycin, methyl (5S,6R)-5-(XP-substituted)-6-alkyl-8-aryl-octa-2E,7E-dienoate (d) where P is a suitable protecting group, using methods known to those of ordinary skill in the art.

One strategy for synthesizing a cryptophycin, which is composed of a S-hydroxy or amino acid unit A, an a-amino acid unit B, a/3-amino acid unit C, and an -hydroxy or amino acid unit D, is to assemble the macrocycle from two precursors representing moieties of the cryptophycin molecule, for example, an A-B precursor (e) containing the 8-hydroxy or amino acid unit A and the a-amino acid unit B and a C-D precursor (f) containing the fl-amino acid unit C and the a-hydroxy or amino acid unit D.
In the method described herein, a cryptophycin is assembled from A-B and C-D
precursors in two steps by (1) connecting the termini of units A and D in the A-B and C-D precursors to form an acyclic C-D-A-B intermediate and (2) connecting the termini of units B and C to form the cyclic product.
In the synthesis of Cryptophycin 51 described in the Experimental Section, an ester linkage is formed between the S-hydroxy group of unit A in the A-B
moiety and the carboxylic acid group of unit D in the C-D fragment to form an acyclic C-D-A-B
intermediate and then an amide linkage is formed between the carboxylic acid group of unit B in the A-B moiety and the (3-amino group of unit C in the C-D moiety.
Compound K is the A-B moiety precursor, Compound P is the C-D moiety precursor, and Compound R is the acyclic C-D-A-B precursor of Cryptophycin 51. Compounds K
and P have protecting groups on the carboxylic acid group of unit B and the )3-amino group of unit C to limit coupling to ester formation between units A and D in step 1.
These protecting groups are removed from the C-D-A-B intermediate so that amide formation can occur between units B and C in step 2.
The synthesis of methyl (5S,6R)-5-t-butyldimethylsilyloxy-6-methyl-8-phenyl-octa-2E,7 E-dienoate (G), the unit A precursor of the Cryptophycin 51, is summarized in Scheme 2 (Figure 6). (S)-trans-3-penten-2-ol (A), the starting material, was prepared by enzymatic resolution of the racemic compound. Reaction of A with propargyl chloride and base under phase-transfer condition formed propargyl ether B in 86% yield.
Treatment of B with butyl lithium at -90 C led to alcohol C in 71 % yield. The desired 3R,4R anti compound C was the only product formed in the Wittig rearrangement.
After protection of the hydroxyl group of C as the tert-butyldimethylsilyl ether (or tert-butyldimethylsilyl ether), hydroboration of the triple bond (H.C. Brown, Organic Synthesis Via Boranes, Wiley, 1975) led to an aldehyde D in 73 % yield from C.
Next D
was converted into the trans a, 16-unsaturated ester E by a Horner-Emmons reaction in 90 % yield. Selective ozonolysis of the C6-C7 double bond in D gave aldehyde F
in 83 %
yield. Finally a Wittig reaction of F with benzyltriphenylphosphonium chloride in the presence of butyllithium produced G in 80 % yield. The yield of G from A was 26 %.
The coupling of the unit A precursor G with the D-3-(3-chloro-4-methoxyphenyl) alanine unit B to produce the A-B precursor (K) is summarized in Scheme 3 (Figure 7).
Hydrolysis of the methyl ester group in G with lithium hydroxide in acetone produced carboxylic acid H in 95 % yield. Coupling of H with trichloroethyl ester I to produce J
could be accomplished in 65 % yield by treating a solution of H in N,N-dimethylformamide (DMF) with a small excess of pentafluorophenyldiphenylphosphinate (FDPP), an equimolar quantity of the trifluoroacetate salt of I, followed by 3 equiv of diisopropylethylamine (DIEA) at 25 C
(S. Chen et al., Tetrahedron Lett. 1991, 32:6711-6714). Fluorodesilylation of J then led to K in 95 % yield.
Protected amino acid I was prepared from D-tyrosine in five steps. First, D-tyrosine was chlorinated with sulfuryl chloride in glacial acetic acid (R.
Zeynek, Hoppe-Seyler's Z. f. Physiol. Chemie 1926, 144:247-254). Next N-(tert-butoxycarbonyl)-3-(3-chloro-4-hydroxyphenyl)-D-alanine was obtained in 94% yield by treating a suspension of the amino acid in 50% aqueous dioxane with di-tert-butyldicarbonate in the presence of triethylamine. The resulting product was dimethylated with dimethyl sulfate in the presence of potassium carbonate in refluxing acetone in 84% yield. The methyl ester was then saponified with sodium hydroxide in aqueous dioxane to yield N-(tert-butoxycarbonyl)-3-(3-chloro-4-methoxyphenyl)-D-alanine in 86 % yield. Exposure of the BOC-protected amino acid to trichloroethanol, pyridine and DCC in dichloromethane led to trichloroethyl ester I in 65 % yield. Treatment of this material with trifluoroacetic acid led to a quantitative yield of the trifluoroacetate salt of I.
The synthesis of (2S)-2-[3'(ten-butoxycarbonyl)amino-2',2'-dimethylpropanoyloxy]-4-methylpentanoic acid (P), the C-D precursor, is summarized in Scheme 4 (Figure 8). The starting point for the unit C portion of P was the aminoalcohol L. Protection of the amino group in L by treatment with di-tert-butyldicarbonate in the presence of triethylamine (93 % yield), followed by oxidation of the primary alcohol with ruthenium tetroxide (P.H.J. Carlsen et al., J. Org. Chem. 1981, 46:3936-3938) gave carboxylic acid M(66 % yield). L-Leucic acid was converted to allyl ester N in 93 %

yield under phase-transfer conditions, by exposing it to a mixture of allyl bromide in dichloromethane and aqueous sodium bicarbonate containing tetra-n-butylammonium chloride (S. Friedrich-Bochnitschek et al., J. Org. Chem. 1989, 54:751-756).
The coupling reaction of M with N was carried out with 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC) in dichloromethane to produce 0 in 75%
yield.
Cleavage of the allyl ester in 0 was carried out in THF containing morpholine and catalytic tetrakis(triphenylphosphine)-palladium to give P in 95% yield (P.D.
Jeffrey et al., J. Org. Chem. 1982, 47:587-590).
Coupling of the A-B precursor (K) and the C-D precursor (P) was accomplished as shown in Scheme 5 (Figure 9). Treatment of K and P with DCC/DMAP in dichloromethane led to the fully protected C-D-A-B intermediate (Q) in 84%
yield.
Reductive cleavage of the trichloroethyl ester group in Q was achieved using activated zinc dust in acetic acid. The BOC-protecting group was then removed by trifluoroacetic acid to give R as the trifluoroacetate salt in 91 % overall yield from Q.
Macrolactamization of R with FDPP led to Cryptophycin 51 in 61 % yield (J.
Dudash, Jr.
et al., Synth. Commun. 1993, 23:349-356). The overall yield from S-trans-3-penten-2-ol (A) was 7%.
Cryptophycin 51 served as the precursor of Cryptophycin 52, the R, R-epoxide, and Cryptophycin 53, the S, S-epoxide. In turn, Cryptophycin 52 served as the precursor of Cryptophycin 55, the 18R,19S-chlorohydrin, and Cryptophycin 57, the 13,14-dihydro analog. Cryptophycin 57 served as the precursor of Cryptophycin 58.
Cryptophycin 53 served as the precursor of Cryptophycin 61 using a method described by T.H.
Chan and J.R. Finkenbine, J. Am. Chem. Soc. 1972, 94:2880-2882 and Cryptophycin 97 using a method described by Y. Ittah et al., J. Org. Chem. 1978, 43:4271-4273.
For the synthesis of cryptophycins that have Ar groups that are different from phenyl, unit A precursors of general structure d (Scheme 1, R3 = Me) can be prepared by a Wittig reaction of aldehyde F (Scheme 2; TBS protecting group) or S
(Scheme 6;
TBPS protecting group) with the appropriate aryltriphenylphosphonium chloride in the presence of butyllithium. Cryptophycin 81 was prepared from precursor d (Ar =
p-methoxyphenyl, R3 = Me) as shown in Schemes 6 and 7 (Figures 10 and 11).
The Ar group can also be introduced into the new cryptophycin at a later step in the synthesis. First precursor d (Ar = R3 = Me) was converted into Cryptophycin 82 by coupling the appropriate A-B (e) and C-D (f) precursors as shown in Scheme 8 (Figure 12). Selective ozonolysis of Cryptophycin 82 or periodic acid oxidation of the corresponding epoxides Cryptophycins 90 and 91 led to an aldehyde, Cryptophycin 108.
} A Wittig reaction of Cryptophycin 108 with the appropriate aryltriphenylphosphonium chloride in the presence of butyllithium gave the new cryptophycin (Scheme 9;
Figure 13). Using this procedure, Cryptophycin 110 (Ar = p-fluorophenyl), Cryptophycin 111 (Ar = p-tolyl), Cryptophycin 112 (Ar = 2-thienyl) and Cryptophycin 124 (Ar = p-chlorophenyl) were prepared. Cryptophycin 110 served as the precursor of the epoxides Cryptophycins 115 and 116. Cryptophycin 111 served as the precursor of the epoxides Cryptophycins 117 and 118 and the chlorohydrins Cryptophycins 128, 130 and 131.
Cryptophycin 112 served as the precursor of the epoxides Cryptophycins 119 and 120.
Cryptophycin 124 served as the precurcur of the epoxides Cryptophycins 125 and 126 and the chlorohydrins Cryptophycins 132, 133 and 134.
Another strategy for synthesizing a cryptophycin is to assemble the macrocycle from three precursors, for example, an A-B precursor (e) containing the 6-hydroxy or amino acid unit A, a precursor containing the 6-hydroxy or amino acid unit D, and a precursor containing the fl-amino acid unit C. In the method described herein, a cryptophycin is assembled from A-B, C and D precursors in three steps by (1) connecting the termini of units A and D in the A-B and D precursors to form an acyclic D-A-B
intermediate, (2) connecting the termini of units D and C in the D-A-B and C
precursors to form an acyclic C-D-A-B intermediate, and (3) connecting the termini of units B and C
to form the cyclic product.
In the synthesis of Cryptophycin 121 described in the Experimental Section, an ester linkage is formed between the 6-hydroxy group of unit A in the A-B
moiety and the carboxylic acid group of unit D in the D fragment to form an acyclic D-A-B
intermediate.
An amide linkage is then formed between the carboxylic acid group of unit C
and the a-amino group of unit D in the D-A-B fragment. Finally an amide linkage is formed between the carboxylic acid group of unit B in the A-B moiety and the 0-amino group of unit C in the C-D moiety. Compound K is the A-B moiety precursor, BOC-L-leucineanhydride is the D unit precursor, and Compound AL is the unit C
precursor.
Compound AK is the acyclic C-D-A-B precursor of Cryptophycin 121 (Scheme 10;
Figure 14). Compounds K and BOC-L-leucineanhydride have protecting groups on the carboxylic acid group of unit B and the a-amino group of unit D to limit coupling to ester formation between units A and D in step 1. The protecting group is removed from the S-amino group of unit D in the D-A-B intermediate so that amide formation can occur between the amino group in unit B and the carboxylic acid C in step 2. These protecting groups are removed from the C-D-A-B intermediate so that amide formation can occur between units B and C in step 2.
Novel cryptophycin compounds of the present invention have epoxide rings that are opened by nucleophiles at different rates than the epoxide ring in Cryptophycin 1, or have chlorohydrin functionalities that form epoxide rings at different rates than the chiorohydrin functionality of Cryptophycin 8 is transformed into the epoxide ring of Cryptophycin 1. The epoxide ring of Cryptophycin 1 or the chlorohydrin functionality (a masked epoxide ring) of Cryptophycin 8 is essential for optimum in vivo activity. If the epoxide oxygen is eliminated (such as found in Cryptophycin 3) or the epoxide ring hydrolyzed to a diol (such as found in Cryptophycin 15), antitumor activity is greatly diminished. Cryptophycin 1 shows appreciable toxicity in animals compared with Cryptophycin 8. This is reflected in the T/C (mostly >0%) and gross log kill values (mostly < 2.0) for Cryptophycin 1 compared with those for Cryptophycin 8 (mostly T/C
values of 0% and gross log kill values of >2.8). Cryptophycin 25, the corresponding bromohydrin analog, shows T/C and gross log kill values that are comparable with those for Cryptophycin 1. This striking difference in in vivo activity suggests that the bromohydrin Cryptophycin 25 is converted more rapidly into Cryptophycin 1 in vivo than Cryptophycin 8. This further suggests that the less toxic Cryptophycin 8 might have more time to accumulate at the tumor site before being transformed into the active compound Cryptophycin 1. 'The epoxide group of Cryptophycin 1 probably binds covalently to its target receptor in the tumor cell. The novel cryptophycins in the present invention could potentially possess better in vivo activity than Cryptophycin 1 and Cryptophycin 8 by exhibiting more favorable rates of epoxide formation in vivo from the corresponding chlorohydrin pro-drugs and covalent binding to the target receptor in the tumor cell.
The compounds of the present invention are more stable towards hydrolysis and solvolysis than Cryptophycins 1 and 21. The ester bond connecting units C and D in Cryptophycin 1 is relatively sensitive to mild base hydrolysis, cleaving at pH
11 to a hydroxy acid with a half-life of 0.83 hour. The C-D ester bond in Cryptophycin 21, which lacks the methyl group on C-2 of unit C, opens at a faster rate with a half-life of 0.25 hour. The C-D ester bond is also sensitive to solvolysis. When methanol is used in the isolation scheme, considerable methanolysis of Cryptophycins 1 and 21 occurs.
Cryptophycin 21 is much more susceptible to methanolysis than Cryptophycin 1.
Cryptophycin 1 shows antitumor activity whereas Cryptophycin 21 is inactive, probably because the C-D ester bond of Cryptophycin 21 is hydrolyzed faster than the C-D ester bond of Cryptophycin 1 in vivo. Hydrolysis of the C-D ester bond may also explain in part the diminished in vivo activity of Cryptophycin 1 by intraperitoneal and subcutaneous routes of drag administration. The C-D ester bond of cryptophycins possessing two methyl groups on C-2 of unit C, such as the one found in Cryptophycin 52, is stable at pH 11.
The compounds of the present invention and the previously disclosed cryptophycin compounds can be therapeutically employed as anti-neoplastic agents and thereby used in methods to treat neoplastic diseases. As used herein, "neoplastic" pertains to a neoplasm, which is an abnormal growth, such growth occurring because of a proliferation of cells not subject to the usual limitations of growth. As used herein, "anti-neoplastic agent" is any compound, composition, admixture, co-mixture or blend which inhibits, eliminates, retards or reverses the neoplastic phenotype of a cell.
Chemotherapy, surgery, radiation therapy, therapy with biologic 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. Chemotherapy utilizing one or more cryptophycins is provided by the present invention. Moreover, combination chemotherapy, chemotherapy utilizing cryptophycins in combination with other neoplastic agents, is also provided by the subject invention as combination therapy is generally more effective than the use of single anti-neoplastic agents. Thus, a further aspect of the present invention provides compositions containing a therapeutically effective amount of at least one new cryptophycin compound of the present invention, including nontoxic addition salts thereof, which serve to provide the above-recited therapeutic benefits. Such compositions can also be provided together with physiologically tolerable liquid, gel or solid carriers, diluents, adjuvants and excipients.

Such carriers, diluents, adjuvants and excipients may be found in the United States Phannacapeia Vol. XXII and National Fotmularv VolXVII, U.S. Phannacopeia Convention, Inc., Rockville, MD (1989).
Additional modes of treatanent are provided in AHFS Drug Infor7nation, 1993 ed. by the American Hospital Formulary Service, pp. 522-660, The present invention further provides that the pharmaceutical composition used to treat neoplastic disease contains at least one cryptophycin compound and at least one additional anti-neoplastic agent. Anti-neoplastic compounds which may be utilized in combination with cryptophycin include those provided in The Merck Index, 11th ed.
Merck & Co., Inc. (1989) pp. Ther 16-17.
In a further embodiment of the invention, anti-neoplastic agents may be antimetabolites which may include, but are not limited to, 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 not limited to, cyclophosphamide, melphalan, busulfan, paraplatin, chlorambucil, and nitrogen mustard. In a further embodiment of the subject invention, the anti-neoplastic agents are plant alkaloids which may include, but are not limited to, vincristine, vinblastine, taxol, and etoposide. In a further embodiment of the present invention, the anti-neoplastic agents contemplated are antibiotics which may include, but are not limited to, doxorubicin (adriamycin), daunorubicin, mitomycin c, and bleomycin. In a further embodiment of the subject inv.ention, the anti-neoplastic agents contemplated are hormones which may include, but are not limited to, calusterone, diomostavolone, propionate, epitiostanol, mepitiostane, testolactone, tamoxifen, polyestradiol phosphate, megesterol acetate, flutamide, nilutamide, and trilotane. In a further embodiment of the subject invention, the anti-neoplastic agents contemplated include enzymes which may include, but are not limited to, L-Asparaginase or aminoacridine derivatives which may include, but are not limited to, amsacrine. Additional anti-neoplastic agents include those provided in Skeel, Roland T., "Antineoplastic Drugs and Biologic Response Modifier:
Classification, Use and Toxicity of Clinically Useful Agents," Handbook of Cancer Chemotherapv (3rd ed.), Little Brown & Co. (1991).

.77_ The present cryptophycin compounds and compositions can be administered to mammals for veterinary use, such as for domestic animals, and clinical use in humans in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particularized requirements of individual hosts. Ordinarily, dosages will range from about 0.001 to 1000mg/kg, more usually 0.01 to 10mg/kg, of the host body weight.
Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time, usually exceeding 24 hours, until the desired therapeutic benefits have been obtained. Indeed, drug dosage, as well as route of administ.ration, must be selected on the basis of relative effectiveness, relative toxicity, growth characteristics of tumor and effect of cryptophycins on cell cycle, drug pharmacokinetics, age, sex, physical condition of the patient, and prior treatment.
The cryptophycin compounds, with or without additional anti-neoplastic agents, may be formulated into therapeutic compositions as natural or salt forms.
Pharmaceutically acceptable non-toxic salts include the base addition salts (formed with free carboxyl or other anionic groups) 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 the further invention provides are those available to one of ordinary skill in the art, for example, that found in the United States Pharnracopeia Vol. XXII and National Formularv Vol XVII, U.S.
Pharmacopeia Convention, Inc., Rockville, MD (1989).
The suitability of particular carriers for inclusion in a given therapeutic composition depends on the preferred route of administration. For example, anti-neoplastic compositions may be formulated for oral administration. Such compositions are typically prepared either as liquid solution or suspensions, or in solid forms. Oral formulations usually include such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and typically contain 1%-95 % of active ingredient, preferably 2%-70%.
Compositions of the present invention may also be prepared as injectable, either as liquid solutions, suspensions, or emulsions; solid forms suitable for solution in, or suspension in, liquid prior to injection may be prepared. Such injectables may be administered subcutaneously, intravenously, intraperitoneally, intramuscularly, intrathecally, or intrapleurally. The active ingredient or ingredients are often mixed with diluents 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 auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.
The invention further provides methods for using cryptophycin compounds encompassed by the genus structure to inhibit the proliferation of mammalian cells by contacting these cells with a cryptophycin compound in an amount sufficient to inhibit the proliferation of the mammalian cell. A preferred embodiment is a method to inhibit the proliferation of hyperproliferative mammalian cells. For purposes of this invention, "hyperproliferative mammalian cells" are mammalian cells which are not subject to the characteristic limitations of growth, e.g., programmed cell death (apoptosis).
A further preferred embodiment is when the mammalian cell is human. The invention further provides contacting the mammalian cell with at least one cryptophycin compound and at least one additional anti-neoplastic agent. The types of anti-neoplastic agents contemplated are the same as those disclosed hereinabove.
The invention fureher provides methods for using cryptophycin compounds encompassed by the genus structure to inhibit the proliferation of hyperproliferative cells with drug-resistant phenotypes, including those with multiple drug-resistant phenotypes, by contacting said cell with a cryptophycin compound 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 the mammalian cell with a cryptophycin compound and at least one additional anti-neoplastic agent. The types of anti-neoplastic agents contemplated are the same as those disclosed hereinabove.
The invention further 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 provided hereinabove to inhibit the proliferation of the hyperproliferating cells. As used herein "pathological condition" refers to any pathology arising from the proliferation of mammalian cells that are not subject to the normal limitations of cell growth. Such proliferation of cells may be due to neoplasms, including, but not limited to the following neoplasms:
mammary, small-cell lung, non-small-cell lung, colorectal, leukemia, melanoma, 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 lymphoma, non-Hodgkin's and Hodgkin's. In a further embodiment of the invention, the neoplastic cells are human. The present invention further provides methods of alleviating such pathological conditions utilizing cryptophycin in combination with other therapies, as well as other anti-neoplastic agents.
Such therapies and their appropriateness for different neoplasia may be found in Cance Principles and Practice of Oncology, 4th ed., Editors DeVita, V., Hellman, S., and Rosenberg., S., Lippincott Co. (1993), In the present disclosure, cryptophycin compounds are shown to potently disrupt the microtubule structure in cultured cells. In-addition, and in contrast with the 1rRca alkaloids, cryptophycin compounds appear to be a poor substrate for the drug-efflux pump P-glycoprotein. Cryptophycin 1 is the major cytotoxin in the blue-green alga (cyanobacteria) Nosioc sp. strain designated GSV 224 and shows excellent activity against tumors implanted in mice. This cyclic didepsipeptide had previously been isolated from Nostoc sp. ATCC accession no. 53789 as an antifungal agent and its gross structure was previously determined. The relative and absolute stereochemistry of this potentially important drug has now been established using a combination of chemical and spectral techniques. Twenty-four additional cryptophycin compounds, Cryptophycins 2-7, 16-19, 21, 23, 24, 26, 28-31, 40, 43, 45, 49, 50 and 54 have also been isolated from and their total structures and cytotoxicities determined. Several derivatives and degradation products are described, both chemically and pharmacologically.
The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
Experimental Section In the experimental disclosure which follows, all weights are given in grams (g), milligrams (mg), micrograms ( g), nanograms (ng), picograms (pg), moles (mol) or millimoles (mmol), all concentrations are given as percent by volume (%), molar (M), millimolar (mM), micromolar ( M), nanomolar (nM), picomolar (pM), or normal (N), all volumes are given in liters (L), milliliters (mL) or microliters ( L), and linear measurements in millimeters (mm), unless otherwise indicated.
The following examples demonstrate the isolation and synthesis of cryptophycin compounds as well as their use as therapeutic agents in accordance with the invention.
In screening extracts of over 1000 blue-green algae (cyanobacteria) for antitumor activity, the lipophilic extract of Nostoc sp. GSV 224 was found to be strongly cytotoxic,3 exhibiting minimum inhibitory concentrations (MICs) of 0.24ng/mL against KB, a human nasopharyngeal carcinoma cell line, and 6ng/mL against LoVo, a human colorectal adenocarcinoma cell line. More importantly, this extract showed significant tumor selective cytotoxicity in the Corbett assay.4=5 Bioassay monitored reversed-phase chromatography of the algal extract led to a fraction which was predominantly Cryptophycin 1, a potent fungicide that had been isolated earlier from Nostoc sp. ATCC
53789 by researchers at Merclc6=' and found to be very active against strains of Cryptococcus.
Cryptophycin 1 accounted for most of the cytotoxic activity of the crude algal extract of Nostoc sp. GSV 224 and the pure compound showed IC50 values of 3 and 5pg/mL against KB and LoVo, respectively. In the Corbett assay Cryptophycin 1 was found to be strongly tumor selective and equally cytotoxic against drug-sensitive and drug-resistant tumor cells. Immunofluorescence assays showed that Cryptophycin interact with a cellular target similar to that of vinblastine, but differed from the latter drug in having a longer time course of action and in not forming paracrystalline bodies.

In preliminary in vivo experiments, Cryptophycin 1 exhibited very promising activity against tumors implanted in mice.
Minor amounts of several other cryptophycin compounds were present in Nostoc sp. GSV 224. Twenty-one of these could be isolated in sufficient quantities for structure determinations and antitumor evaluation in vitro by extraction of the alga with 1 : 5 dichloromethane/acetonitrile and reversed-phase HPLC of the extract. .
Cryptophycins 2, 3, 4, 16, 17, 18, 19, 21, 23, 24, 26, 28, 29, 30, 31, 40, 43, 45, 49, 50 and accompanied Cryptophycin 1 in the fraction eluted from a reversed-phase flash column with 65:35 acetonitrile/water. Cryptophycins 2, 3, 4, 5, 6, and 7 were the only compounds found when the alga was extracted with methanol and the reversed-phase chromatography was carried out with methanol/water. Cryptophycins 2, 3, 4, 5 and 6 were eluted with 3:1 methanol/water and Cryptophycin 7 was found in an earlier, less cytotoxic fraction eluted with 1:3 methanol/water. Acyclic Cryptophycins 5, 6 and 7 appear to be artifacts generated by decomposition of Cryptophycin 1 during the isolation procedure.
Cryptophycins 3 and 5 appeared to be identical with fungicidal semi-synthetic compounds prepared from Cryptophycin 1 by researchers at Merck.8=9 Cryptophycin 3 was prepared by treating Cryptophycin 1 with a zinc-copper couple or with diphosphorus tetraiodide.8 Cryptophycin 5 was prepared by methanolysis of Cryptophycin 1.9 Examnle 1 Structure Determination The determination of the structures of the new compounds, viz. Cryptophycins 2, 4, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 21, 23, 24, 26, 28, 29, 30, 31, 40, 43, 45, 49, 50 and 54, as well as those previously disclosed, were carried out in a straightforward manner using methodology that is well-known to those trained in the art. Mass spectral data were consistent with the molecular compositions. Proton and carbon NMR
data obtained from COSY, HMQC, HMBC and NOESY spectra allowed one to assemble all of the gross structures of these depsipeptide-type compounds. The presence of the various hydroxy and amino acid units in each compound were confirmed by gas chromatographic mass spectral analysis. Total structures, including absolute stereochemistries, were determined using a combination of chemical degradative and special analytical techniques on appropriate derivatives of the cryptophycin compounds.

Example 2 Structure-Activity Relationships (SAR) To probe the structural features in Cryptophycin 1 needed for optimal activity, all of the compounds described herein were evaluated for cytotoxicity against KB
(human nasopharyngeal carcinoma), LoVo (human colon carcinoma), and SKOV3 (human ovarian carcinoma) cell lines. IC50 values are listed in Tables 1 and 2. Comparison of the cytotoxicities show that the intact macrolide ring, the epoxy and methyl groups and the double bond in the 7,8-epoxy-5-hydroxy-6-methyl-8-phenyl-2-octenoic acid unit (see unit A in Figure 1), the chloro and 0-methyl groups in the 3-(3-chloro-4-methoxyphenyl) alanine unit (unit B), the methyl group in the 3-amino-2-methylpropionic acid unit (unit C), and the isobutyl group in the leucic acid unit (unit D) of Cryptophycin 1 are needed for optimal cytotoxicity. The potent cytotoxicity of Cryptophycin 8 is most likely due to the chlorohydrin functionality which acts as a masked epoxide.
The most active compounds were also evaluated for selective cytotoxicity against four different cell types, viz. a murine leukemia (L1210 or P388), a murine solid tumor (colon adenocarcinoma 38, pancreatic ductal adenocarcinoma 03, mammary adenocarcinoma M16/M17), a human solid tumor (colon CX-1, HCT8, H116; lung H125;
mammary MX-1, MCF-7), and a low malignancy fibroblast (LML), using the Corbett assay4, a disk diffusion assay modeled after the one commonly used in antifungal and antibacterial testing. The results, shown in Table 1, indicated that Cryptophycins 1-5 and 8 were neither solid tumor nor leukemia selective, but rather equally active against tumor cell lines, including drug-resistant ones such as M17. None of the compounds showed a zone of inhibition for any of the solid tumor cell lines that was 250 zone units, i.e.
7.5mm, larger than the zone of inhibition for the leukemia cell line.
Cryptophycins 1-5 and 8, however, displayed markedly larger zones of inhibition (400 zone units) for all of the tumor cell lines compared with the zone of inhibition for the fibroblast LML.
Diagnostically LML has been found to behave more like a normal cell than a tumor cell with respect to clinically-useful cytotoxic agents (see Corbett assay data for fluorouracil, etoposide and taxol in Table 1). Since the differential cytotoxicities were > 250 zone units, Cryptophycins 1-5 and 8 were tumor selective. These compounds therefore became candidates for in vivo testing.
Cryptophycin 1 is active against a broad spectrum of murine and human tumors implanted in mice, including drug-resistant ones (Table 3). It exhibits excellent activity against five early stage murine tumors, viz. colon adenocarcinomas #38 and #51, taxol-sensitive and taxol-resistant mammary #16/C/RP, and pancreatic ductal adenocarcinoma #03, and two early stage human tumors tested in SCID mice, viz. MX-1 breast and H125 adenosquamous lung, showing tumor burden T/C (mean tumor burden in treated animals/mean tumor burden untreated animals) values that are less than 10%.
T/C values that are less than 42% are considered to be active by NCI
standards;
T/C values that are less than 10% are considered to have excellent activity and potential clinical activity by NCI standards.4 Two of the trials showed gross (tumor cell) log kill values of 2Ø Gross log kill is defmed as T-03.2 Td where T is the median tiune in days for the tumors of the treated group to reach 750mg, C is the median time in days for the tumors of the control group to reach 750mg, and Td is the tumor volume doubling time. Gross log kill values of >2.8, 2.0-2.8, 1.3-1.9, 0.5-0.8, and <0.5 with duration of drug treatment of 5-20 days are scored + + + +, + + +, + +, + and -(inactive), respectively. An activity rating of +++ to ++++, which is indicative of clinical activity, is needed to effect partial or complete regression of 100-300mg size masses of most transplanted solid tumors of mice.
Cryptophycin 8 is also active against a broad spectrum of tumors implanted in mice (Table 4). It has shown excellent activity against all of the tumors tested to date, showing tumor burden T/C values < 10%, but more importantly gross log kill activity ratings of +++ to ++++ and some cures.
Good in vivo activity was also seen with Cryptophycin 35 in the one trial that has been run to date.

Lethal toxicity observed during testing of Cryptophycins 1 and 8 was attributed to leucopenia which is common to all clinically used antitumor drugs.

Example 3 Culture Conditions Nostoc sp. GSV 224 was obtained from Professor C.P. Wolk, MSU-DOE Plant Research Laboratory, Michigan State University. Nostoc sp. ATCC 53789 was purchased from the American Type Culture Collection. A 1L flask culture of alga was used to inoculate an autoclaved 20L glass carboy containing an inorganic medium, designated modified BG-113, the pH of which had been adjusted to 7.0 with NaOH. Cultures were continuously illuminated at an incident intensity of 200 mol photons m-Zsec' (photosynthetically active radiation) from banks of cool-white fluorescent tubes and aerated at a rate of 5L/min with a mixture of 0.5 % CO2 in air at a temperature of 24 t 1' C. Typically, the culture was harvested by filtration after 21 days. The yields of lyophilized Nostoc sp. GSV 224 and ATCC 53789 averaged 0.61 and 0.3g/L of culture, respectively.

Example 4 Isolation Method A: The lyophilized Nostoc sp. GSV224 (50g) was extracted with 2 L of 1:

CH2C12/CH3CN for 48 h and the extract concentrated in vacuo to give a dark green solid.
The residue (1g; KB MIC 0.24ng/mL) was applied to an ODS-coated silica column (55g, 7 x 5cm) and subjected to flash chromatography with 1:3 CH3CN/H20 (0.8 L), 1:1 CH3CN/H20 (0.8 L), 65:35 CH3CN/H20 (1.0 L), MeOH (0.8 L), and CH2C12 (0.5 L).
The fraction that was eluted with 65:35 CH3CN/H20 (420mg; KB MIC 14pg/mL) was subjected to reversed-phase HPLC (Econosil C18, 10 , 25cm x 21.5mm, UV
detection at 250nm, 65:35 CH3CN/H2O, flow rate 6mL/min) to obtain Cryptophycin 1(tR 49.3 min, 220mg) and a number of impure fractions. The fraction eluted from the Econosil column at tR 28.8 min was further purified by normal phase HPLC (Econosil silica 51t cartridge, 250 x 4.6mm, 6:4 ethyl acetate/hexane, 3mL/min) to give Cryptophycin 16 (3.0mg). The fraction eluted from the Econosil C18 column at tR 32.5 min was subjected to HPLC on the Econosil silica column using 55:45 ethyl acetate/hexane at 3mL/min to give Cryptophycin 24 (0.8mg). The fraction eluted from the Econosil C18 column at tR
35.5 min was subjected to HPLC twice on the Econosil silica column, first using 1: 1 ethyl acetate/hexane at 3mL/min and second using 4 : 6 ethyl acetate /methylene chloride at 2.5mL/min to give Cryptophycin 23 (1.2mg) and Cryptophycin 43 (0.1mg). The fraction eluted from the Econosil C18 column at tR 39.5 min was subjected to HPLC on the Econosil silica column with 1:1 ethyl acetate/hexane at 3mL/min to give Cryptophycin 2 (6mg) and Cryptophycin 21 (14mg) and a complex mixture of cryptophycins eluted at tR
32.5 min. This latter fraction, accumulated from 400g dry alga, was chromatographed successively on a semi preparative column (partisil C18, 250 x 9.4mm, l01L) with 35:65 water/acetonitrile and a reversed phase analytical column (Econosil, 150 x 4.6mm, 5 ) with 5:4:1 water/acetonitrile/methanol at 1.3mL/min to give Cryptophycin 50 (tR 34.8, 0.4mg) and Cryptophycin 40 (tR 38.8 min, 0.3mg). The fraction eluted from the Econosil C18 column at tR 44.5 min was subjected to HPLC on the Econosil silica column with 1:1 ethyl acetate/hexane at 3mL/min to give Cryptophycin 17 (0.3mg). Normal phase HPLC
purification of the fraction eluted from the Econosil C18 column at tR 54.5 as a shoulder to Cryptophycin 1 yielded Cryptophycin 45 (tR 6.7 min, 0.1mg), Cryptophycin 26 (tR 8.9 min, 0.5mg), and Cryptophycin 54 (tR 19.8 min, <0.1mg) on elution with 1:1 ethyl acetate/hexane. The fraction eluted from the Econosil C18 column as a broad peak (tR 58 to 70 min) was subjected to HPLC on the Econosil silica colunm with 43:57 ethyl acetate/hexane at 2.5mL/min to give Cryptophycin 4(tR 19.6 min, 1.5mg), Cryptophycin 31 (tR 9.4 min, 0.8mg), Cryptophycin 19 (tR 25.8min, 0.3mg), Cryptophycin 49 (tR 28 min, 0.lmg), Cryptophycin 28 (tR 29.0min, 0.5mg) and impure Cryptophycin 29 (tR 52.5 min, 2.0mg) and Cryptophycin 30 (tR 49 min, 3.0mg). Cryptophycins 29 and 30 obtained pure after reversed phase HPLC (Econosil C18, 10 , 250 x 10mm, 3:1 methanol/water). The fraction eluted from the Econosil C18 column at tR 78.9 min was subjected to HPLC

on the Econosil silica column with to give Cryptophycin 3(tR 16.4 min, 3.0mg).
The fraction eluted from the Econosil C18 column at tR 82.8 min was subjected to HPLC on the Econosil silica column with 45:55 ethyl acetate/hexane at 3mL/min to give Cryptophycin 18 (tR 19.2, 0.8mg).
Method B: The lyophilized Nostoc sp. GSV 224 (12.23g) was extracted twice with 700mL and 400mL portions of MeOH for 12 and 5 hours (h), respectively. The extracts were combined and concentrated in vacuo to give 1.84g of a dark green solid which was partitioned between water and CH2C12. The lipophilic portion (0.65g; KB MIC
0.24ng/mL) was applied to an ODS-coated silica column (55g, 7 x 5cm) and subjected to flash chromatography with 1:3 MeOH/H20 (0.8L), 1:1 MeOH/H20 (0.8L), 3:1 MeOH/H2O (0.8L), MeOH (0.8L), and CH2C12 (0.5L). The fraction that was eluted with 3:1 MeOH/H20 (22mg; KB MIC 14pg/mL), which accounted for essentially all of the cytotoxic activity, was subjected to reversed-phase HPLC (Econosil C18, 10 , x10mm, UV detection at 250nm, flow rate 3mL/min) using 1:5 MeOH/H20 as the eluant to give Cryptophycins 7(tR 7.6 min, 0.2mg), 5(tR 15.4 min, 2.3mg), 2(tR 16.0 min, 1.0mg), 1(tR 19.0 min, 12.0mg), 4(tR 26.5 min, 1.2mg), and 3(tR 30.2 min, 1.4mg).
From one of the cultures the fraction (8.lmg) that eluted from the flash column with 1:3 MeOH/H20 showed milder cytotoxicity (KB MIC 2 g/mL). Purification on HPLC
using 2:3 MeOH/H2O as the eluant yielded Cryptophycin G (7, tR 6.0 min, 2.4mg).

Example 5 Spectral Data for Cryptophycins 1-7 The bold italicized letters in the spectral data refer to the units A-D in Figure 1.
Cryptophycin 1 [a]D +33.8' (MeOH, c 1.83); UV X.(e) 208 (42,400), 218 (33,700), 228 (23,800), 280 (2,210); CD [0]202 +15,900, [0]206 +64,900, [0]214 +26,900, [0]224 +46,300, [0]237 +10,500. IR (CHC13) P. 3425, 2963, 1751, 1719, 1677, 1502, cm'. EIMS m/z (rel intensity) 654/656 (20/9), 412/414 (33/12), 280/282 (31/12), 227 (80), 195/197 (92/44), 91 (100); high resolution EIMS m/z 654.2665 (calcd for C35H43C1N208, 4.3mmu error). 'H NMR (CDC13): amino or hydroxy acid unit S(carbon position, multiplicity; J in Hz) 7, 8-epoxy-5-hydroxy-6-methyl-8 phenyl-2-octenoic acid (A) 5.74 (2, dt; 15.5 and 0.9), 6.68 (3, ddd; 15.5, 9.6 and 5.2), 2.45 (4, ddd;
14.2, 11.1 and 9.6), 2.55 (4, brdd; 14.2 and 5.2), 5.16 (5, ddd; 11.1, 4.9 and 1.9), 1.80 (6, m), 1.14 (6-Me, d; 7.1), 2.92 (7, dd; 7.5 and 2.0), 3.69 (8, d; 2.0), 7.25 (10/14, m), 7.34-7.39 (11/12/13, m); leucic acid (D) 4.83 (2, dd; 6.8 and 3.3), 1.70 (3, m), 1.36 (3, m), 1.70(4, m), 0.86 (5, d; 6.6), 0.85 (4-Me, d; 6.6); 3-amino-2-methylpropionic acid (C) 2.71 (2, m), 1.22 (2-Me, d; 7.1), 3.30 (3, ddd; 13.4, 5.8 and 3.8), 3.48 (3, ddd; 13.4, 6.3 and 5.8), 6.93 (3-NH, brt; 5.8); 3-chloro-4-methoxyphenylalanfne (B) 4.80 (2, ddd;
8.7, 7.3 and 5.4), 5.61 (2-NH, d; 8.7), 3.03 (3, dd; 14.4 and 7.3), 3.13 (3, dd; 14.4 and 5.4), 7.21 (5, d; 2.1), 3.87 (7-OCH3,s), 6.83 (8, d; 8.5), 7.07 (9, dd; 8.5 and 2.1). 13C
NMR (CDC13): unit 6(carbon position) A 165.3 (1), 125.3 (2), 141.0 (3), 36.7 (4), 76.2 (5), 40.6 (6), 13.5 (6-Me), 63.0 (7), 59.0 (8), 136.7 (9), 125.6 (10/14), 128.7 (11/13), 128.5 (12); D 170.7 (1), 71.3 (2), 39.4 (3),.24.5 (4), 22.9 (5), 21.3 (4-Me);
C 175.6(1), 38.2 (2), 14.1 (2-Me), 41.1 (3); B 170.9 (1), 53.6 (2), 35.0 (3), 129.7 (4), 131.0 (5), 122.4 (6), 154.0 (7), 56.1 (7-OCH3), 112.2 (8), 128.4 (9).
Cryptophycin 2 [a]D +20.4' (MeOH, c 0.54); UV X.(e) 206 (43,800), 218 (37,500), 232 (22,900), 278 (2,410); CD [0]203 +54,100, [0]212 +16,500, [0]=5 +53,600, [0]236 -14,000. IR (CHC13) P. 3423, 3029, 2961, 1742, 1724, 1678, 1512, 1258 cm-'.
EIMS
m/z (rel intensity, assignment) 620 (11, M*), 431 (3), 378(8), 377 (6), 311 (11), 246 (10), 244 (8), 227 (14), 195 (17), 161 (84, CH3O-C6H4-CH=CH=CO+), 121 (79, C6H4-CHZ+), 91 (100); high resolution EIMS m/z 620.3094 (calcd for C35H44N2O8, 0.3mmu error); 161.0605 (calcd for C10I1902, -0.2mmu error); 121.0658 (calcd for C8H9O, -0.4mmu error). 'H NMR (CDC13): amino or hydroxy acid unit S(carbon position, multiplicity; J in Hz) 7, 8-epoxy-5-hydroxy-6-methyl-8 phenyl-2-octenoic acid (A) 5.71 (2, dd; 15.4 and 1.3), 6.70 (3, ddd; 15.4, 10.2 and 5.0), 2.45 (4, m), 2.55 (4, m), 5.18 (5, ddd; 11.3, 4.8 and 2.0), 1.79 (6, m), 1.14 (6-Me, d; 7.0), 2.92 (7, dd; 7.7 and 2.0), 3.68 (8, d; 2.0), 7.24 (10/14, m), 7.34-7.39 (11/12/13, m); leucic acid (D) 4.82 (2, dd; 10.1 and 3.7), 1.70 (3, m), 1.33 (3, m), 1.70 (4, m), 0.86 (5, d; 6.4), 0.84 (4-Me, d; 6.4); 3-amino-2-methylpropionic acid (C) 2.68 (2, m), 1.23 (2-Me, d; 7.3), 3.39 (3-H2, m), 7.02 (3-NH,brt; 6.0); O-methyltyrosine (B) 4.79 (2, ddd; 8.1, 7.0 and 5.7), 5.55 (2-NH, d; 8.1), 3.07 (3, dd; 14.5 and 7.0), 3.13 (3, dd; 14.5 and 5.7), 7.10 (5/9, d;
8.6), 6.81 (6/8, d; 8.6), 3.78 (7-OCH3, s). 13C NMR (CDC13): unit S(carbon position) A
165.1 (1), 125.1 (2), 141.1 (3), 36.7 (4), 76.0 (5), 40.7(6), 13.6 (6-Me), 63.0 (7), 59.0 (8), 136.7 (9), 125.6 (10/14), 128.7 (11/13), 128.5 (12); D 170.6(1), 71.3 (2), 39.4 (3), 24.5 (4), 21.3 (5), 22.9 (4-Me); C 176.0 (1), 38.1 (2), 14.2 (2-Me), 40.7 (3);
B 171.1 (1), 53.9 (2), 35.3 (3), 131.0 (4), 130.2 (5/9), 114.1 (6/8), 158.6 (7), 55.2 (7-OCH3).
Crxptophycin 3 [a]D +20.3 (MeOH, c 1. 13); UV X.(e) 206 (51,700), 218 (31,200), 230 (22,900), 246 (18,800), 280 (3,230); CD [0]205 +50,000, [0]212 -390, [0]218 -47,200, [61233 -100, [8]25l +33,400, [0]271 +4,310. IR (CHC13) v,,. 3417, 2926, 1742, 1721, 1676, 1499, 1336 cm 1. EIMS m/z (rel intensity) 638/640 (2/0.7, M+), 412/414 (63/19), 280/282 (15/5), 227 (100), 195 (63), 91 (98); high resolution EIMS m/z 638.2764 (calcd for C35H43C1N207, -0.5mmu error), 412.1516 (calcd for C20H27CIN06, 1.lmmu error), 227.1293 (calcd for C15H17NO, 1.Ommu error). 'H NMR (CDC13): amino or hydroxy acid unit 8(carbon position, multiplicity; J in Hz) 5-hydroxy-6-methyl-8 phenyl-2, 7-octadienoic acid (A) 5.77 (2, d; 15.5), 6.68 (3, ddd; 15.5, 9.5 and 5.3), 2.37 (4, m), 2.54 (4, m), 5.01 (5, ddd; 11.4, 6 and 1.5), 2.56 (6, m), 1.14 (6-Me, d; 7.0), 6.01 (7, dd; 15.8 and 8.8), 6.41 (8, d; 15.8), 7.28-7.34 (10/11/13/14, m), 7.23 (12, m); leucic acid (D) 4.84 (2, dd; 10.1 and 3.6), 1.62 (3, m), 1.36 (3, m), 1.62 (4, m), 0.77 (5, d;
6.5), 0.73 (4-Me, d; 6.3); 3-amino-2-methylpropionic acid (C) 2.71 (2, m), 1.22 (2-Me, d; 7.3), 3.28 (3, dt; 13.5 and 7.0), 3.50 (3, ddd; 13.5, 4.9 and 4), 6.93 (3-NH, brt; 6.3);
3-chloro-4-methoxyphenylalanine (B) 4.82 (2, m), 5.64 (2-NH, d; 8.8), 3.05 (3, dd; 14.5 and 7.0), 3.13 (3, dd; 14.5 and 5.5), 7.22 (5, d; 2.2), 3.87 (7-OCH3, s), 6.84 (8, d;
8.5), 7.08 (9, dd; 8.5 and 2.2). 13C NMR (CDC13): unit 8(carbon position) A
165.4 (1), 125.2 (2), 141.4 (3), 36.5 (4), 77.1 (5), 42.3 (6), 17.3 (6-Me), 130.1(7), 130.0 (8), 136.7 (9), 126.1 (10/14), 128.6 (11/13), 128.4 (12); D 170.1 (1), 71.6 (2), 39.5 (3), 24.5 (4), 21.2 (5), 22.7 (4-Me); C 175.6 (1), 38.3 (2), 14.0 (2-Me), 41.2 (3);
B 170.9 (1), 53.5 (2), 35.1 (3), 129.8 (4), 131.0 (5), 122.4 (6), 154.0 (7), 56.1 (7-OCH3), 112.2 (8), 127.6 (9).
Cryptophycin 4 [a]D +36.7 (MeOH, c 1.93); UV) A.(e) 206 (41,800), 228 (25,000), 240 (21,200), 248 (22,500), 280 (3,000), 290 (1,230); CD [0]205 +63,900, [0]211 +3,040, [e1218 -71,900, [0]229 -11,700, [0]234 -130,[01u2 +47,500, [91270 +5,400. IR
(CHC13)v.
3410, 2962, 2917, 1741, 1718, 1678, 1511, 1251 cm-'. EIMS mlz (rel intensity) 604 (2, M+), 378 (74), 246 (11), 227 (46), 161 (100), 91 (96); high resolution EIMS
m/z 604.3127 (calcd for C35H44N2O7, 2.2mmu error), 378.1910 (calcd for C20H28NO6, 0.7mmu error), 227.1293 (calcd for C15H17NO, 1.7mmu error), 161.0605 (calcd for C,oH902, -0.2mmu error). 'H NMR (CDC13): amino or hydroxy acid unit 8(carbon position, multiplicity; J in Hz) 5-hydroxy-6-methyl-8 phenyl-2, 7-octadienoic acid (A) 5.74 (2, dd;
15.3 and 1.2), 6.71 (3, ddd; 15.3, 10.3 and 5.0), 2.37 (4, m), 2.53 (4, m), 5.03 (5, ddd;
11.2, 6.4 and 2.0), 2.55 (6, m), 1.13 (6-Me, d; 6.8), 6.01 (7, dd; 15.8 and 8.8), 6.40 (8, d; 15.8), 7.28-7.37 (10/11/13/14, m), 7.22 (12, m); leucic acid (D) 4.84 (2, dd; 10.1 and 3.6), 1.65 (3, m), 1.34 (3, m), 1.65 (4, m), 0.75 (5, d; 6.5), 0.72 (4-Me, d;
6.3); 3-amino-2-methylpropionic acid (C) 2.69 (2, m), 1.22 (2-Me, d; 7.5), 3.39 (3-H2, m), 7.03 (3-NH, brt; 6.0); O-methyltyrosine (B) 4.79 (2, m), 5.61 (2-NH, d; 7.8), 3.08 (3, dd;
14.5 and 7.0), 3.13 (3, dd; 14.5 and 5.3), 7.11 (5/9, d; 8.8), 6.81 (6/8, d;
8.8), 3.78 (7-OCH3, s). 13C NMR (CDC13): unit S(carbon position) A 165.3 (1), 125.1 (2), 141.5 (3), 36.5 (4), 77.1 (5), 42.3 (6), 17.3 (6-Me), 130.1 (7), 131.8 (8), 136.7 (9), 126.2 (10/14), 128.7 (11/13), 127.6 (12); D 170.8 (1), 71.6 (2), 39.5 (3), 24.5 (4), 21.2 (5), 22.7 (4-Me); C 175.9 (1), 38.2 (2), 14.2 (2-Me), 40.9 (3); B 171.2 (1), 53.8 (2), 35.3 (3), 131.0 (4), 130.2 (5/9), 114.1 (6/8), 158.6 (7), 55.2 (7-OCH3).
CrXptophycin 5 [a]D +36.0' (MeOH, c 0.55); UV X.(e) 206 (45,600), 218 (37,700), 280 (3,790), 286 (3,480), 325 (2,080); CD [0]203 +7,710, [9]206 +29,000, [0]210 +21,400, [0]= +59,800, [0]234 +12,800, [0124, +13,700. IR (CHC13) v,,. 3426, 2958, 1728, 1672, 1502, 1259 cm'. EIMS m/z (rel intensity) 686/688 (0.1510.05), 655/657 (1/0.3), 654/656 (1.5/0.5), 311/313 (75/27), 195 (66), 155 (54), 121 (51), 91 (100);
high resolution EIMS m/z 686.2983 (calcd for C36H47CIN209, -1.3mmu error). 'H NMR
(CDC13): amino or hydroxy acid unit 6(carbon position, multiplicity; J in Hz) 7, 8-epoxy-S-hydroxy-6-methyl-8 phenyl-2-octenoic acid (A) 5.87 (2, d; 15.3), 6.72 (3, dt; 15.3 and 6.8), 2.60 (4, m), 2.52 (4, ddd; 15.2, 7.8, and 6.8), 5.11 (5, ddd; 12.3, 7.8, and 7.1), 1.87 (6,m), 1.12 (6-Me, d; 7.1), 2.91 (7, dd; 7.3 and 2.1), 3.70 (8, d; 2.1), 7.24 (10/14, brd; 7.4), 7.29-7.36 (11/12/13, m); leucic acid (D) 4.09 (2, m), 2.86 (2-OH, brd, 6.1), 1.83 (3, m), 1.42 (3, m), '1.86 (4, m), 0.90 (5, d; 6.6), 0.87 (4-Me, d; 6.8);
3-amino-2-methylpropionic acid (C) 3.64 (1-OCH3, s), 2.60 (2, m), 1.07 (2-Me, d; 7.3), 3.27 (3, ddd; 13.5, 8.0 and 5.5), 3.39 (3, m), 6.32 (3-NH, t; 5.4); 3-chloro-4-methoxyphenylalanine (B) 4.59 (2, dt; 6 and 7.5), 6.30 (2-NH, d; 7.5), 2.95 (3, dd; 13.6 and 7.5), 3.0 (3, dd; 13.6 and 6.0), 7.2 (5, d; 2.1), 3.86 (7-OCH3, s), 6.84 (8, d; 8.5), 7.05 (9, dd, 8.5; 2.1). 13C NMR (CDC13): unit 6(carbon position) A 164.8 (1), 126.5 (2), 139.2 (3), 34.4 (4), 75.5 (5), 39.2 (6), 12.9 (6-Me), 63.3 (7), 58.7 (8), 136.8 (9), 125.7 (10/14), 128.6 (11/13), 128.4 (12); D 175.1 (1), 69.2 (2), 43.2 (3), 24.3 (4), 21.2 (5), 23.2 (4-Me); C 175.4 (1), 51.9 (1-OMe), 39.1 (2), 14.7 (2-Me), 41.6 (3);
B 170.6 (1), 54.6 (2), 37.4 (3), 129.5 (4), 131.0 (5), 122.4 (6), 154.1 (7), 56.1 (7-OMe), 112.2 (8), 128.4 (9).
Cryptophycin 6 [a]D +17.1 (MeOH, c 1.1); UV X,.(e) 206 (40,000), 218 (30,100), 228 (21,400), 282 (2,430); CD [0]203 +37,700, [01210 -5,430, [0]213 -1,260, [0]221 +24,100, [6]232 +8,480, [9]2 +13,400, [0]254 +790. IR (CHC13) v11187C 3425, 3006, 2956, 1726, 1672, 1641, 1502, 1462, 1259 cm 1. FABMS (thioglycerol) m/z, (rel intensity) (13/6) [M-H2O]+, 217 (26), 91 (100). 'H NMR(CDC13): amino or hydroxy acid unit S
(carbon position, multiplicity; J in Hz) 5, 7, 8-trihydroxy-6-methyl-8 phenyl-2-octenoic acid (A) 5.92 (2, dt; 15.0 and 1.5), 6.94 (3, dt; 15 and 7.5), 2.51 (4, m), 2.64 (4, m), 3.97 (5, ddd; 9.3, 6.5 and 4.5), 2.03 (6, m), 1.10 (6-Me, d; 6.5), 3.70 (7, dd; 9.0 and 7.5), 4.64 (8, d; 7.5), 7.33-7.39 (10/11/13/14, m), 7.28 (12, tt; 6.5 and 2.0); 3-chloro-4-methoxyphenylalanine (B) 4.60 (2, td; 8.0 and 6.0), 6.09 (2-NH, brd; 8.0), 2.96 (3, dd;
.30 13.8 and 8.0), 3.02 (3, dd; 13.8 and 6.0), 7.22 (5, d; 2.0), 3.86 (7-OCH3, s), 6.84 (8, d;
8.5), 7.07 (9, dd; 8.5 and 2.0) 3-amino-2-methylpropionic acid (C) 3.63 (1-OCH3,s), 2.58 (2, m), 1.07 (2-Me, d; 7.0), 3.24 (3, ddd; 13.8, 8 and 6.5), 3.41 (3, ddd; 13.8, 6.5 and 4.8), 6.21 (3-NH, brt; 6.5). 13C NMR (CDC13): unit 6(carbon position) A
165.2 (1), 125.6 (2), 141.3 (3), 36.9 (4), 82.5 (5), 46.3 (6), 14.3 (6-Me), 85.1 (7), 84.8 (8), 140.9 (9), 125.8 (10/14), 128.6 (11/13), 127.8 (12); B 170.6 (1), 54.5 (2), 37.3 (3), 129.6 (4), 131.0 (5), 122.5 (6), 154.1 (7), 56.1 (7-OCH3), 112.2 (8), 128.5 (9) C 52.0 (1-OCH3), 175.4 (1), 39.2 (2), 14.7 (2-Me), 41.6 (3).
Cryptoph cy in 7 [a]D -51.9 (MeOH, c 0.89); UV X.(e) 206 (23,400), 220 (14,900), 282 (1,670);
CD [0]202 +35,400, je1206 -1,730, 101211 -19,200, [0]220 -15,800, [0]232 +29,000, [0]263 +2,040. IR (CHC13) P. 3426, 2946, 1732, 1675, 1501, 1258 cm-1. EIMS m/z (rel intensity) 455/457 (1/0.3, [M-2HZO]+), 105 (100), 77 (98); FABMS m/z (magic bullet matrix) 496/498 [M-H20+Na]+, (thioglycerol matrix) 474/476 [M-H201+H]+. 'H NMR
(CD3OD): amino or hydroxy acid unit a(carbon position, multiplicity; J in Hz) 5, 7, 8-trihydroxy-6-methyl-8 phenyl-2-octenoic acid (A) 6.06 (2, ddd; 15.5, 1.3 and 1.0), 6.80 (3, dt; 15.5 and 7.5), 2.49 (4, m), 2.59 (4, m), 3.92 (5, ddd; 9.5, 6.3 and 4.7), 1.95 (6, m), 1.08 (6-Me, d; 6.7), 3.59 (7, dd; 9.0 and 7.8), 4.56 (8, d; 7.8), 7.37 (10/14, brd;
7.3), 7.31 (11/13, brt; 7.3), 7.24 (12, tt; 7.3 and 1.5); 3-chloro-4-methoxyphenylalanfne (B) 4.52 (2, dd; 6.9 and 5.0), 2.93 (3, dd; 13.8 and 6.9), 3.15 (3, dd; 13.8 and 5.0), 7.20 (5, d; 2.2), 3.78 (7-OCH3, s), 6.88 (8, d; 8.4), 7.08 (9, dd; 8.4 and 2.2). 13C
NMR (CD3OD): unit S(carbon position) A 167.4 (1), 127.6 (2), 140.9 (3), 37.9 (4), 84.0 (5), 47.6 (6), 14.4 (6-Me), 86.0 (7), 85.8 (8), 142.9 (9), 127.1 (10/14), 129.3 (11/13), 128.5 (12); B 177.6 (1), 57.3 (2), 38.2 (3), 132.8 (4), 132.1 (5), 122.9 (6), 155.0 (7), 56.5 (7-OCH3), 113.2 (8), 130.1 (9).
Cryptophycin 16 [a]D + 41.3 (MeOH, c 5.2); UV ~, (e) 242 (4963), 280 (2430), 286 (2212); IR
(neat) v11187C 3402, 3270, 2960, 1748, 1724, 1676, 1514, 1466, 1343, 1239, 1177 cm-1;
EIMS m/z (rel intensity) 640/642 (66/27), 398/400 (47/16), 265 (55), 227 (93), (100); high resolution EIMS m/z 640.25676 (calcd for C34H41C1N2O8, -1.6mmu error).
'H NMR (CDC13): amino or hydroxyacid unit 6(carbon position, multiplicity; J
in Hz) 7, 8-epoxy-5-hydroxy-6-methyl-8-phenyl-2-octenoic acid (A) 5.74 (2, d; 16), 6.67 (3, ddd;
15.3, 9.7 and 5.5), 2.45 (4, dt; 14.3 and 10.4), 2.55 (4, brdd; 14.3 and 5.3), 5.15 (5, ddd; 11.2, 4.8 and 1.8), 1.8 (6, m), 1.14 (6-Me, d; 7.0), 2.92 (7, dd; 7.5 and 2.0), 3.69 (8, d; 2.0), 7.24-7.26 (10/14, m), 7.33-7.39 (11/12/13, m); 3-chloro-4-hydroxyphenylalanine (B) 4.8 (2, m), 5.64 (2-NH, d; 8.8), 3.03 (3, dd; 14.5 and 7.0), 3.11 (3, dd; 14.4 and 5.6), 7.17 (5, d; 2.2), 5.61(7-OH, s), 6.91 (8, d; 8.3), 7.0 (9, dd;
8.3 and 2.2); 3-amino-2-methylpropionic acid (C) 2.71 (2, m), 1.22 (2-Me, d;
7.3), 3.28 (3, dt; 13.6 and 6.8), 3.49 (3, ddd; 13.6, 5 and 4.1), 6.92 (3-NH, br t; 6.1);
leucic acid (D) 4.83 (2, dd; 10.1 and 3.3), 1.36 (3, m), 1.67-1.75 (3, m), 1.67-1.75 (4, m), 0.85 (5, d; 7.5), 0.86 (4-Me, d; 6.8). 13C NMR (CDC13) unit 6(carbon position) A 165.3 (1), 125.3 (2), 141.0 (3), 36.7 (4), 76.2 (5), 40.6 (6), 13.5 (6-Me), 63.0 (7), 59.0 (8), 136.8 (9), 125.6 (10/14), 128.7 (11/13), 128.6 (12); B 170.9 (1), 53.6 (2), 35.1 (3), 129.9 (4), 129.6 (5), 120.0 (6), 150.4 (7), 116.4 (8), 129.2 (9); C 175.6 (1), 38.3 (2), 14.1 (2-Me), 41.1 (3); D 170.8 (1), 71.3 (2), 39.4 (3), 24.6 (4), 21.3 (5), 22.9 (4-Me).
Cryptophvcin 17 [a]D + 27.8 (CHC13 c. 0.37); UV ~(e) 248 (14740), 268 (8100), 278 (3400), 284 (2840); IR (neat) v11187C 3412, 2958, 1750, 1723, 1668, 1504, 1463, 1290, 1177, 751 cm-'; EIMS m/z (rel intensity) 624/626 (10/3), 398/400 (95/35), 284 (100), 149 (95);
high resolution EIMS m/z 624.26161 (calcd for C34H41C1NZO7, -1.4mmu error). 'H
NMR
(CDC13): amino or hydroxyacid unit 6(carbon position, multiplicity; J in Hz) 5-hydroxy-6-methyl-8 phenyl-2, 7-octadienoic acid (A) 5.77 (2, d; 15.4), 6.67 (3, ddd;
15.4, 9.5, and 5.3), 2.37 (4, m), 4.99 (5, ddd; 11.2, 6.3, and 1.6), 2.54 (6, m), 1.14 (6-Me, d; 6.7), 6.01 (7, dd; 15.7, and 8.7), 6.41 (8, d; 15.9), 7.28-7.34 (10/11/13/14, m), 7.23 (12, m);
3-chloro-4-hydroxyphenylalanine (B) 4.82 (2, m), 5.63 (2-NH, d; 8.7), 3.12 (3, dd; 14.7, and 5.6), 3.03 (3', dd; 14.7, and 7.1), 7.18 (5, d; 2.0), 5.47 (7-OH, br s), 6.91 (8, d;
8.3), 7.02 (9, dd; 8.3, and 2.0); 3-amino-2-methylpropionic acid (C) 2.71 (2, m), 1.21 (2-Me, d' 6.9), 3.25 (3, m), 3.52 (3', m), 6.89 (3-NH, br t; 6.1); luecic acid (D) 4.84 (2, dd; 9.6, and 3.1), 1.62 (3, m), 1.36 (3', m), 1.62 (4, m), 0.77 (5, d' 6.5), 0.73 (4-Me, d; 6.5); 13C NMR (CDC13) unit 6(carbon position) A 165.4 (1), 125.3 (2), 141.3 (3), 36.5 (4), 77.1 (5), 42.3 (6), 17.3 (6-Me), 130.0 (7), 129.9 (8), 136.7 (9), 126.2 (10/14), 128.6 (11/13), 127.6 (12); B 170.9 (1), 53.5 (2), 35.1 (3), 129.6 (4), 131.9 (5), 126.2 (6), 150.3 (7), 116.3 (8), 127.6 (9); C 175.9 (1), 38.4 (2), 13.9 (2-Me), 41.3 (3);
D 170.9 (1), 71.6 (2), 39.5 (3), 24.5 (4), 21.2 (5), 22.7 (4-Me).

Cryptophvcin 18 [a]D + 54.9 (MeOH, c 0.93); UV A. (e) 250 (20518), 284 (3857); IR (neat) P. 3411, 3271, 2966, 1746, 1728, 1668, 1505, 1463, 1258, 1178 cm-1; EIMS m/z (rel intensity) 638/640 (4.5/1.1), 412/414 (59/19), 280(17), 227 (100); high resolution EIMS
m/z 638.272934 (calcd for C35H43C1N207, 2.9mmu error). 'H NMR (CDC13): amino or hydroxy acid unit S(carbon position, multiplicity; J in Hz) 5-hydroxy-6-methyl-8 phenyl-2, 7-octadienoic acid (A) 5.76 (2, d; 15.5), 6.65 (3, ddd; 15.4, 9.2 and 6.2), 2.38-2.47 (4, m), 5.08 (5,ddd; 10.6, 4.9 and 2.2), 2.58 (6, m), 1.15 (6-Me, d; 6.8), 6.07 (7, dd;
15.9 and 8.5), 6.43 (8, d; 15.9), 7.21-7.35 (10/11/12/13/14, m); 3-chloro-4-methoxy-phenylalanine (B) 4.83 (2, m), 3.05(3, dd; 14.5 and 7.1), 5.65 (2-NH, d; 8.7), 3.14 (3, dd; 14.4 and 5.5), 7.21 (5, d; 2.4), 3.86 (7-OCH31 s), 6.83 (8, d; 8.3), 7.08 (9, dd; 8.3 and 2.2); 3-amino-2-methylpropionic acid (C) 2.73 (2, m), 1.23 (2-Me, d; 7.2), 3.23 (3, dt; 13.5 and 6.8), 3.56 (3, ddd; 13.5, 5.7 and 4.0), 6.85 (3-NH, dd; 7.1 and 6.2); leucic acid (D) 4.8 (2, d; 4.6), 1.86-1.89 (3, m), 0.94 (3-Me, d; 7.0), 1.20-1.26 (4, m), 1.39-1.44 (4, m), 0.77 (5, d; 7.4). 13C NMR (CDC13) unit 8(carbon position) A 165.5 (1), 125.2 (2), 141.5 (3), 36.4 (4), 77.7 (5), 41.9 (6), 17.1 (6-Me), 129.8 (7), 131.9 (8), 136.8 (9), 128.6 (10/14), 126.2 (11/13), 127.6 (12); B 170.0 (1), 53.5 (2), 35.1 (3), 129.9 (4), 131.1 (5), 122.4 (6), 153.9 (7), 56.1 (7-OCH3), 112.2 (8), 128.5 (9); C 175.3 (1), 38.6 (2), 14.0 (2-Me), 41.4 (3); D 169.5 (1), 76.6 (2), 36.2 (3), 15.5 (3-Me), 24.2 (4), 14.0 (5).
Cryptophvcin 19 [aJD +62.6 (MeOH, c 0.67); UV (MeOH) X. (e) 204 (44900), 230 (17000), 248 (15600), 280 (2500); IR (neat) v,,m 3413, 3272, 2966, 1745, 1726, 1672, 1504, 1258, 1199, 1178, 1066, 692 cm'; EIMS m/z (rel intensity) 624/626 (3.0/1.4), (58/21), 280/282(15/5), 227 (100), 195/197 (57/22); high resolution EIMS m/z 624.2585 (calcd for C34H41C1N2O7, 1.8mmu error). 'H-NMR (CDC13):amino or hydroxy acid unit 6 (carbon position, multiplicity; J in Hz) 5-hydroxy-6-methyl-8 phenyl-2, 7-octadienoic acid (A) 5.76 (2, d; 15.2), 6.64 (3, ddd; 15.4, 9.1 and 6.2), 2.38 (4, m), 2.47 (4, m), 5.04 (5, ddd;7.1, 5.1 and 1.8), 2.57 (6, m), 1.15 (6-Me, d; 6.9), 6.05 (7, dd; 15.8 and 8.5), 6.43 (8, d; 15.8), 7.29-7.35 (10/11/13/14, m), 7.23 (12, m); 3-chloro-4-methoxyphenylalanine (B) 4.84 (2, m), 5.67 (2-NH, d; 8.9), 3.04(3, dd; 14.3 and 7.1), 3.14 (3, dd; 14.3 and 5.3), 7.22 (5, d; 2.0), 3.86 (7-OCH3, s), 6.83 (8, d;
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~0-60-L661 S9SbiZZO Va (64), 215/217/219 (31/20/8), 141 (100); high resolution EIMS m/z 674.21643 (calcd. for C34H4C12N2O8, -0.3mmu error); 1H NMR (CDC13) amino or hydroxyacid unit b(carbon position, multiplicity; J in Hz) 7, 8-epoxy-5-hydroxy-6-methyl-8 phenyl-2-octenoic acid (A) 5.77 (2, d; 15.4), 6.65 (3, ddd; 15.4, 9.3 and 6.0), 2.47 (4, dt; 14.2 and 10.2), 2.55 (4, br dd; 14.2 and 5.6), 5.13 (5, ddd; 11.0, 4.6 and 1.6), 1.81 (6, m), 1.15 (6-Me, d;
6.9), 2.93 (7, dd; 7.6 and 2.0), 3.7 (8, d; 2.0), 7.22-7.26 (10/14, m); 7.32-7.39 (11/12/13, m); 3,5-dichloro-4-hydroxyphenylalanine (B) 4.81 (2, m), 5.69 (2-NH, d;
8.6), 3.11 (3, dd; 14.5 and 5.6), 3.50 (3, dd; 14.3 and 7.0), 7.13 (5/9, s), 5.78 (7-OH, s); 3-amino-2-methylpropionic acid (C) 2.73 (2, m), 1.22 (2-Me, d; 7.1), 3.19 (3, dt;
13.4 and 6.9), 3.58 (3, ddd; 13.6, 5.8 and 4.1), 6.82 (3-NH, br t; 5.9);
leucic acid (D) 4.84 (2, dd; 9.9 and 3.2), 1.38 (3, m), 1.68-1.75 (3, m), 1.68-1.75 (4, m), 0.86 (4-Me, d; 6.7), 0.87 (5, d; 6.7). 13C NMR (CDC13) unit S(carbon position) A 165.4 (1), 125.4 (2), 140.9 (3), 36.7 (4), 76.3 (5), 40.6 (6), 13.5 (6-Me), 63.0 (7), 58.9 (8), 136.7 (9), 125.6 (10/14), 128.7 (11/13), 128.6 (12); B 170.7 (1), 53.3 (2), 35.0 (3), 130.3 (4), 129.0 (5/9), 121.0 (6/8), 146.7 (7); C 175.3 (1), 38.4 (2), 13.9 (2-Me), 41.5 (3); D
170.8 (1), 71.3 (2), 39.4 (3), 24.6 (4), 21.3 (4-Me), 22.9 (5).
Cryptophvcin 24 [a]D + 48.8 (CHC13, c 0.63); UV A,,. (e) 228 (19006), 242 (8249), 274 (2351);
IR (neat) v,,,a~ 3400, 3284, 2959, 1732, 1678, 1652, 1514, 1248, 1178 cm 1;
EIMS m/z (rel intensity, assignment) 606 (2, M+), 364 (7), 161 (55, CH3O-C6H4-CH=CH=CO+), 121 (100, CH3O-C6H4-CH2+), 91 (68); high resolution EIMS m/z 606.2954 (calcd for C34H42N2O8, -1.3mmu error); 1H NMR (CDC13) amino or hydroxy acid unit S(carbon position, multiplicity; J in Hz) 7, 8-epoxy-5-hydroxy-6-methyl-8 phenyl-2-octenoic acid (A) 5.70 (2, dd; 15.2 and 1.3), 6.70 (3, ddd; 15.2, 10.3 and 4.7), 2.43 (4, dt;
14.3 and 10.9), 2.56 (4, m), 5.20 (5, ddd; 11.3, 5.1 and 2.0), 1.79 (6, m), 1.14 (6-Me, d; 7.0), 2.92 (7, dd; 7.5 and 2.0), 3.68 (8, d; 2.0), 7.23 -7.38 (10/11/12/13/14, m); 0-methyl-tyrosine (B) 4.73 (2, m), 5.58 (2-NH, d; 8.3), 3.03 (3, dd; 14.5 and 7.5), 3.14 (3, dd;
14.5 and 5.7), 7.11 (5/9, d; 8.6), 6.81 (6/8, d; 8.6), 3.78 (7-OMe, s); 3-aminopropionic acid (C) 2.55 (2-H2, m), 3.42 (3, m), 3.53(3, m), 6.97 (3-NH, br t; 5.7);
leucic acid (D) 4.89 (2, dd; 9.9 and 3.5), 1.29 (3, m), 1.62 - 1.70 (3/4, m), 0.83 (5, d;
5.9), 0.84 (4-Me, d; 6.1); 13C NMR (CDC13): unit 6(carbon position) A 165.4 (1), 125.3 (2), 141.0 (3), 36.7 (4), 75.9 (5), 40.6 (6), 13.4 (6-Me), 63.0 (7), 59.0 (8), 136.7 (9), 125.6 (10/14), 128.7 (11/13), 128.5 (12); B 170.7 or 170.6 (1), 54.1 (2), 35.2 (3), 128.5 (4), 130.2 (5/9), 114.1 (6/8), 158.6 (7), 55.2 (7-OMe); C 172.8 (1), 32.5 (2), 34.2 (3); D
170.6 or 170.7 (1), 71.2 (2), 39.5 (3), 24.4 (4), 21.3 (5), 22.8 (4-Me).
Cryptophycin 26 [a]D + 28.2 (CHC13, c 1.31); UV A. (e) 254 (14615), 284 (2949); IR (neat) v1187C 3299, 2960, 1732, 1644, 1504, 1258, 1209 cm 1; EIMS m/z (rel intensity) (0.5/0.1, M+), 638/640 (1.7/1.0), 525/527 (3.7/1.8), 412/414 (10/4), 280/282 (12/11), 227 (20), 195 (48), 131 (68); high resolution EIMS m/z 656.2836 (calcd for C35H45C1N208, 2.8mmu error), 638.2712 (calcd for C35H43C1N207, 4.7mmu error);
iH
NMR (CDC13) amino or hydroxy acid unit 6(carbon position, multiplicity; J in Hz) 3, 5-dihydroxy-6-methyl-8 phenyl-7-octenoic acid (A) 2.46 (2, dd; 14.8 and 7.8), 2.58 (2, dd;
14.8 and 3.0), 5.46 (3, m), 1.86 - 1.90 (4-H2, m), 3.61 (5, m), 2.37 (6, m), 1.14 (6-Me, d; 6.8), 6.06 (7, dd; 16 and 8.7), 6.47 (8, d; 16), 7.37 (10/14, br d; 7.9), 7.32 (11/13, br t; 7.6), 7.22 - 7.28 (12, m); 3-chloro-4-methoxyphenylalan ine (B) 4.73 (2, br dt; 6.4 and 8.1), 6.14 (2-NH, d; 8.6), 2.84 (3, dd; 14.4 and 8), 3.18 (3, dd; 14.4 and 6.3), 7.21 (5, d; 2.2), 3.85 (7-OMe, s), 6.82 (8, d; 8.6), 7.08 (9, dd; 8.6 and 2.2); 3-amino-2-methylpropionic acid (C) 2.87 (2, m), 1.19 (2-Me, d; 7.0), 3.01 (3, ddd; 13.4, 10.6 and 4.9), 3.73 (3, ddd; 13.4, 8.2 and 4.7), 6.72 (3-NH, br dd; 7.3 and 5.2);
leucic acid (D) 4.95 (2, dd; 9.7 and 4.2), 1.62 - 1.72 (3, m), 1.79 - 1.84 (3, m), 1.62 - 1.72 (4, m), 0.90 (4-Me, d; 6.4), 0.95 (5, d; 6.4). 13C NMR (CDC13): unit 6(carbon position) A
170.0 (1), 41.5 (2), 71.4 (3), 37.3 (4), 71.9 or 71.8 (5), 43.6 (6), 16.6 (6-Me), 130.8 (7), 132.5 (8), 136.8 (9), 126.2 (10/14), 128.6 (11/13), 127.6 (12); B 170.9 (1), 53.2 (2), 34.7 (3), 130.3 (4), 131.1 (5), 122.2 (6), 153.8 (7), 56.1 (7-OMe), 112.2 (8), 128.5 (9); C 174.3 (1), 40.1 (2), 14.4 (2-Me), 42.5 (3); D 170.7 (1), 71.8 or 71.9 (2), 38.9 (3), 24.6 (4), 21.6 (4-Me), 22.9 (5).
Cryptovhcyin 28 [a]D + 65.6 (MeOH, c 0.93); UV (MeOH) X. (e) 204 (48000), 230 (19300), 248 (18700), 280 (3400); IR (neat) v. 3413, 3270, 2958, 1745, 1726, 1665, 1504, 1258, 1197, 1175, 1066, 694 cm'; EIMS m/z (rel intensity) 624/626 (3.0/1.3), (70/24), 280/282(13/6), 213 (100), 195/197 (86/40); high resolution EIMS m/z 624.2626 (calcd for C34H41C1N2O7, -2.4mmu error); 1H NMR(CDC13) amino or hydroxy acid unit 6 (carbon position, multiplicity; J in Hz) 5-hydroxy-8 phenyl-2, 7-octadienoic acid (A) 5.78 '(L) 6'~ST '(9) b'ZZI '(S) 0'I~T '41) 6'6ZI '(~) 6'V~ '(Z) 8'~S '(1) 6'0Li S'=(ZT) 9'LZT
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~0-60-L661 S9SbiZZO Va WO 96/40184 PCT/[JS96/03246 56.1 (7-OMe), 112.2 (8), 128.4 (9); C 172.6 (1), 32.4 (2), 34.5 (3); D 170.4 (1), 71.5 (2), 39.7 (3), 24.4 (4), 21.2 (4-Me), 22.6 (5).
Cryptophycin 30 [a]D - 12.3 (CHC13, c 1.53); UV X. (e) 254 (17200), 284 (3600); IR (neat) v,,=
3414, 3306, 2961, 1738, 1729, 1660, 1504, 1258, 1205, 1183, 1066, 695 cni 1;
EIMS
m/z (rel intensity) 656/658 (1.0/0.3), 638/640 (3.0/1.0), 525/527 (3.8/1.3), (10.5/3.6), 280/282(10.3/3.8), 227 (29), 195/197 (48/17), 155/157 (74/21), 131 (100);
high resolution EIMS m/z 656.2852 (calcd for C35H45C1N208, 1.3mmu error); 1H-NMR
(CDC13) amino or hydroxy acid unit S(carbon position, multiplicity; J in Hz) 3,5-dihydroxy-6-methyl-8-phenyl-7-octenoic acid (A) 2.25 (2, dd; 16.0 and 9.6), 2.64 (2, brd;
16.0), 3.89 (3, m), 2.51 (3-OH, d; 6.4), 1.77 (4, ddd; 14.3, 9.8 and 2.1), 1.88 (4, ddd;
14.3, 11.3 and 3.8), 4.88 (5, ddd; 11.3, 6.2 and 2.1), 2.53 (6, m), 1.10 (6-Me, d; 6.8), 5.99 (7, dd; 15.9 and 9.0), 6.40 (8, d; 15.9), 7.28-7.33 (10/11/13/14, m), 7.23 (12, m);
3-chloro-4-methoxyphenylalanine (B) 4.60 (2, m), 6.61 (2-NH, d; 8.1), 3.09 (3, dd; 14.2 and 5.6), 3.15 (3, dd; 14.2 and 7.3), 7.22 (5, d; 2.1), 3.86 (7-OMe, s), 6.83 (8, d; 8.3), 7.07 (9, dd; 8.3 and 2.1); 3-amino-2-methylpropionic acid (C) 2.67 (2, m), 1.21 (2-Me, d; 7.3), 3.26 (3, ddd; 13.6, 7.3 and 6.4), 3.63 (3, ddd; 13.6, 6.2 and 3.9), 6.75 (3-NH, br t; 6.3); leucic acid (D) 4.83 (2, dd; 9.6, 4.1), 1.42 (3, m), 1.64 (3, m), 1.64 (4, m).
0.79 (4-Me, d; 6.4), 0.76 (5, d; 6.4); 13C NMR (CDC13) unit S(carbon position) A 171.6 (1), 42.4 (2), 66.0 (3), 41.3 (4), 76.0 (5), 42.0 (6), 17.3 (6-Me), 130.0 (7), 131.9 (8), 136.7 (9), 126.1 (10/14), 128.6 (11/13), 127.6 (12); B 170.8 (1), 54.3 (2), 35.1 (3), 130.1 (4), 131.1 (5), 122.2 (6), 153.8 (7), 56.1 (7-OMe), 112.1 (8), 128.7 (9); C 175.6 (1), 39.7 (2), 13.8 (2-Me), 41.5 (3), D 171.9 (1), 72.1 (2), 39.1 3), 24.6 (4), 21.4 (4-Me), 22.7 (5).
Cryptophvcin 31 ja]D + 50.6 (MeOH, c 1.13); UV ~(e) 242 (3800), 284 (700); IR (neat) v.
3412, 3272, 2961, 1745, 1725, 1678, 1537, 1481, 1270, 1196, 1176, 1000, 698 cm 1;
EIMS m/z (rel intensity) 688/690/692 (1.2/1.0/0.4), 446/448/450 (7.9/6.7/3.1), 314/316/318 (17/11/3), 91 (100); high resolution EIMS m/z 688.2336 (calcd for 30 C35H42C12N208, -1.8mmu error); 'H-NMR (CDC13) amino or hydroxy acid unit 8(carbon position, multiplicity; J in Hz) 7, 8-epoxy-S-hydroxy-6-methyl-8 phenyl-2-octenoic acid (A) 5.78 (2, d; 15.5), 6.66 (3, ddd; 15.5, 9.4 and 6.0), 2.47 (4, ddd; 14.1, 10.8 and 9.4), 2.56 (4, m), 5.14 (5, ddd; 10.8, 4.7 and 1.7), 1.82 (6, m), 1.15 (6-Me, d;
7.1), 2.93 (7, dd; 7.5 and 1.9), 3.70 (8, d; 1.9), 7.24-7.26 (10/14, m), 7.34-7.39 (11/12/13, m); 3,5-dichloro-4-methoxyphenylalanine (B) 4.83 (2, m), 5.68 (2-NH, d; 9.0), 3.0 (3, dd; 14.4 and 7.3), 3.14 (3, dd; 14.4 and 5.6), 7.16 (5/9, s), 3.87 (7-OMe, s); 3-amino-methylpropionic acid (C) 2.74 (2, m), 1.22 (2-Me, d; 7.1), 3.20 (3, m), 3.58 (3, ddd;
13.5, 5.6 and 4.1), 6.82 (3-NH, br t; 5.6); leucic acid (D) 4.83 (2, m), 1.38 (3, m), 1.72 (3, m), 1.72 (4, m). 0.87 (4-Me, d; 6.8), 0.86 (5, d; 6.8); 13C NMR (CDC13) unit S
(carbon position) A 165.4 (1), 125.4 (2), 141.0 (3), 36.7 (4), 76.3 (5), 40.6 (6), 13.5 (6-Me), 63.0 (7), 58.9 (8), 136.7 (9), 125.6 (10/14), 128.7 (11/13), 128.6 (12);
B 170.8 (1), 53.3 (2), 35.2 (3), 129.3 (4), 129.6 (5/9), 134.5 (6/8), 151.2 (7), 60.6 (7-OMe); C
175.3 (1), 38.3 (2), 13.9 (2-CH3), 41.5 (3), D 170.6 (1), 71.3 (2), 39.4 (3), 24.6 (4), 22.9 (4-Me), 21.3 (5).
Cryptophycin 40 [a]D + 41.6 (CHC13, c 0.31); UV X,,. (e) 242 (4974), 266 (3911), 274 (3666), 286 (2359), 328 (511); IR (neat) P. 3415, 2959, 1748, 1723, 1667, 1505, 1463, 1289, 1176 cm-'; EIMS m/z (rel intensity) 640/642 (5/2), 280/282 (7/3), 213 (13), (51/17), 155 (29), 141 (32), 121 (28), 91 (100), 69 (47); high resolution EIMS
m/z 640.2570 (calcd. for C34H41C1N2O8, -1.8mmu error); 1H NMR (CDC13) amino or hydroxy acid unit 8(carbon positions, multiplicities; J in Hz) 7, 8-epoxy-S-hydroxy-8 phenyl-2-octenoic acid (A) 5.77 (2, d; 15.1), 6.72 (3, ddd; 15.1, 9.7 and 4.9), 2.42 (4, m), 2.58 (4, m), 5.33 (5, m), 1.89 (6, ddd; 12.9, 8.1 and 5.0), 2.13 (6, ddd; 12.9, 9.3 and 5.0), 2.98 (7, ddd; 6.7, 4.5 and 1.9), 3.64 (8, d; 1.9), 7.31-7.39 (10/11/13/14, m), 7.22 (12, m); 3-chloro-4-methoxyphenylalanine (B) 4.83 (2, m), 5.64 (2-NH, d; 8.6), 3.03 (3, dd;
14.3 and 7.5), 3.14 (3, dd; 14.3 and 5.4), 7.21 (5, d; 2.0), 3.87 (7-OMe, s), 6.84 (8, d;
8.3), 7.08 (9, dd; 8.3 and 2.0); 3-amino-2-methylpropionic acid (C) 2.72 (2, m), 1.23 (2-Me, d; 7.3), 3.31 (3, dt; 13.8 and 6.9), 3.50 (3, ddd; 13.6, 5.7 and 3.9), 6.96 (3-NH, br t; 6.0); leucic acid (D) 4.85 (2, dd; 6.7, 3.4), 1.42 (3, m), 1.72 (3, m), 1.72 (4, m), 0.86 (4-Me, d, 3.7), 0.87 (5, d, 3.7); 13C NMR (CDC13) unit S(carbon position) A 165.3 (1), 125.2 (2), 140.9 (3), 39.0 (4), 72.0 (5), 37.3 (6), 59.0 (7), 58.7 (8), 140.9 (9), 125.6 (10/14), 128.7 (11/13), 128.5 (12); B 170.9 (1), 53.6 (2), 35.1(3), 129.8 (4), 131.0 (5), 122.5 (6), 157.0 (7), 56.1 (7-OMe), 112.3 (8), 128.4 (9); C 175.6 (1), 38.3 (2), 14.1 (2-Me), 41.1 (3); D 170.9 (1), 71.4 (2), 39.4 (3), 24.5 (4), 21.5 (4-Me), 22.8 (5).
Cryptophycin 43 [ca]D + 20' (CHC13, c 0.2); UV X,,. (e) 250 (20512), 282 (4083), 294 (1734);
IR
(neat) P. 3400, 3272, 2927, 1727, 1660, 1516, 1455, 1242, 1175 cm'; EIMS m/z (rel intensity) 533 (24), 484 (3), 445 (14), 398 (9), 364 (29), 227 (59), 149 (67), 91 (100) high resolution EIMS m/z 590.3044 (calcd for C34H41N207, -5.2mmu error); 1H
NMR
(CDC13) amino or hydroxy acid unit 8(carbon position, multiplicity; J in Hz) 5-hydroxy-6-methyl-8 phenyl-2, 7-octadienoic acid (A) 5.75 (2, d; 15.3), 6.69 (3, ddd;
15.3, 9.9 and 5.3), 2.37 (4, dt; 14.2 and 10.4), 2.52 (4, m), 5.01 (5, ddd; 11.2, 6.4 and 1.8), 2.55 (6, m), 1.13 (6-Me, d; 6.9), 6.01 (7, dd; 15.8 and 8.9), 6.41 (8, d; 15.8), 7.21-7.34 (10/11/12/13/14, m); 4-methoxyphenylalanine (B) 4.80 (2, m), 5.64 (2-NH, d;
8.4), 3.06(3, dd; 14.5 and 7.2), 3.13 (3, dd; 14.4 and 5.3), 7.06 (5/9, d; 8.4), 6.74 (6/8, d;
8.4); 3-amino-2-methylpropionic acid (C) 2.69 (2, m), 1.22 (2-Me, d; 7.3), 3.33.(3, m), 3.44 (3, dt; 14.0 and 4.7), 7.0 (3-NH, m); leucic acid (D) 4.84 (2, dd; 10.0 and 3.6), 1.60-1.67 (3, m), 1.35 (3, m), 1.60-1.67 (4, m), 0.76 (5, d; 6.4), 0.73 (4-Me, d; 6.7);
13C NMR (CDC13) unit 6(carbon position) A 125.2 (2), 141.5 (3), 36.5 (4), 77.5 (5), 42.3 (6), 17.3 (6-Me), 130.1 (7), 131.8 (8), 136.8 (9), 126.2 (10/14), 128.6 (11/13), 127.6 (12); B 53.8 (2), 35.3 (3), 129.8 (4), 130.5 (5/9), 115.6 (6/8), 154.6 (7); C 38.3 (2), 14.1 (2-Me), 41.0 (3); D 71.6 (2), 39.6 (3), 24.5 (4), 21.2 (5), 22.9 (4-Me). Due to the small sample size, carbonyl carbon signals could not be seen.
Cryptophvcin 45 [a]D + 72.0 (MeOH, c 0.12); UV X. (e) 250 (25500), 284 (5300); IR (neat) P. 3407, 3239, 2958, 1743, 1727, 1667, 1538, 1469, 1242, 1196, 1177, 694 cm 1;
EIMS m/z (rel intensity) 658/660/662 (2.1/1.4/0.3), 483 (7.6) 432/434/436 (9.5/6.4/1.8), 300/302/304 (8.0/5.5/1.2), 227 (100) 91 (87); high resolution EIMS m/z 658.2207 (calcd for C341-I,oC12N2O7, 0.6mmu error); 'H NMR (CDC13):amino or hydroxy acid unit (carbon position, multiplicity; J in Hz) 5-hydroxy-6-methyl-8 phenyl-2, 7-octadienoic acid (A) 5.80 (2, d; 14.7), 6.66 (3, ddd; 14.7, 8.5 and 5.5), 2.38 (4, m), 2.53 (4, m), 4.97 (5, br dd; 10.4 and 6.2), 2.57 (6, m), 1.14 (6-Me, d; 6.7), 6.01 (7, dd; 15.9 and 8.7), 6.42 (8, d; 15.9), 7.28-7.34 (10/11/1314, m), 7.22 (12; m); 3,5-dichloro-4-hydroxyphenyl-alanine (B) 4.82 (2, m), 5.73 (2-NH, br d; 8.7), 3.02 (3, dd; 14.3 and 6.2), 3.10 (3, dd; 14.3 and 5.2), 7.14 (5/9, s), 5.79 (7-OH, s); 3-amino-2-WO 96/40184 PCT/US96/0324.6 methylpropionic acid (C) 2.73 (2, m), 1.21 (2-Me, d; 7.0), 3.17 (3, m), 3.60 (3, m), 6.81 (3-NH, br t; 6.7); leucic acid (D) 4.84 (2, dd;10.0 and 3.2), 1.38 (3, ddd; 14.9, 10.2 and 3.2), 1.65 (3, m), 1.65 (4, m). 0.78 (4-Me, d; 6.5), 0.73 (5, d;
6.5); 13C NMR
(CDC13) unit 8(carbon position) A 165.5 (1), 125.4 (2), 141.2 (3), 36.4 (4), 77.6 (5), 42.3 (6), 17.3 (6-Me), 130.0 (7), 131.9 (8), 136.7 (9), 126.2 (10/14), 128.6 (11/13), 127.6 (12); B 171.0 (1), 53.2 (2), 35.0 (3), 130.4 (4), 129.1 (5/9), 121.0 (6/8), 146.7 (7); C 175.2 (1), 38.5 (2), 13.9 (2-Me), 41.6 (3), D 170.7 (1), 71.5 (2), 39.5 (3), 24.6 (4), 22.7 (4-Me), 21.2 (5).
Cryptophycin 49 [a]D +68.1 (MeOH, c 0.075); W X. (e) 246 (25500), 284 (5200); IR (neat) v.. 3401, 3282, 2962, 1744, 1728, 1668, 1540, 1505, 1464, 1258, 1198, 1177, 1066, 694 cm''; EIMS m/z (rel intensity) 624/626 (0.8/0.3), 398/400 (43/14), 227(78), 195/197 (58/26) 91 (100); high resolution EIMS m/z 624.2650 (calcd for C34H41C1NZO7, -4.8mmu error); 'H NMR(CDC13):amino or hydroxy acid unit 6(carbon position, multiplicity; J in Hz) 5-hydroxy-6-methyl-8 phenyl-2, 7-octadienoic acid (A) 5.77 (2, d; 14.1), 6.67 (3, m), 2.38 (4, m), 2.50 (4, m), 5.01 (5, m), 2.56 (6, m), 1.13 (6-Me, d; 6.5), 6.03 (7, dd;
15.8 and 8.6), 6.42 (8, d; 15.8), 7.29-7.35 (10/11/13/14, m), 7.23 (12; m); 3-chloro-4-methoxyphenylalanine (B) 4.82 (2, m), 5.64 (2-NH, m), 3.06 (3, m), 3.13 (3, m), 7.22 (5, m), 3.87 (7-OMe, s), 6.83 (8, m), 7.08 (9, m); 3-amino-2-methylpropionic acid (C) 2.72 (2, m), 1.22 (2-Me, d; 6.7), 3.26 (3, m), 3.53 (3, m), 6.90 (3-NH, m); 2-hydroxyvaleric acid (D) 4.81 (2, dd; 8.8 and 3.9), 1.63 (3, m), 1.68 (3, m), 1.33 (4-H2, m). 0.74 (5, t; 7.3).
Cryptophycin 50 [a]D + 32.0 (CHC13 c. 0.44); UV A. (e) 242 (4933), 262 (3996, 274 (3719), 286 (2430), 332 (359); IR (neat) v. 3412, 3274, 2958, 1752, 1724, 1676, 1648, 1503, 1465, 1258, 1177, 1066, 753; EIMS m/z (rel intensity) 640/642 (4/2), 398/400 (11/4), 280/282 (10/3), 227 (17), 195/197 (57/18), 157 (20), 141 (31), 91 (100); high resolution EIMS m/z 640.2531 (calcd. for C34H41C1N2O8, 2.lmmu error); 'H NMR (CDC13) amino or hydroxy acid unit 8(carbon positions, multiplicities; J in Hz) 7, 8-epoxy-5-hydroxy-6-methyl-8-phenyl octanoic acid (A) 5.73 (2, d; 15.7), 6.67 (3, ddd; 15.7, 9.7 and 5.4), 2.45 (4, m), 2.55 (4, m), 5.13 (5, ddd; 11.2, 5.0 and 1.7), 1.78 (6, m), 1.15 (6-Me, d, 6.9), 2.91 (7, dd; 7.5 and 1.9), 3.68 (8, d; 1.7), 7.25 (10/14, m), 7.33-7.38 (11/12/13;

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~0-60-L661 S9SbiZZO Va Example 6 Synthesis of Cr~Utophycin Derivatives Cryptonhvcin 8 To a solution of 3.8mg of Cryptophycin 1 in 1.5mL of 2:1 1,2-dimethoxyethane/water was added 91tL 1N HCI. The solution was allowed to stir at room tempierature for 4 h, neutralized with potassium carbonate, and evaporated.
The residue was partitioned between water and CHZC12. The CHZC12-soluble material was purified by reversed-phase HPLC to obtain 3.3mg of pure Cryptophycin 8.
EIMS m/z (relative intensity) 690/692/694 (0.8/0.5/0.2). High resolution EIMS
m/z 690.2533 (calcd for C35H44C12N2O8, -5.8mmu error). 'H NMR (CDC13): amino or hydroxy acid unit S(carbon position, multiplicity; J in Hz) 8-chloro-5, 7-dihydroxy-6-methyl-8 phenyl-2-octenoic acid (A) 5.79 (2, d; 15.4), 6.69 (3, ddd; 15.4, 9.7 and 5.6), 2.68 (4, ddt; 14.0, 5.5 and 1.8), 2.38 (4,m), 5.11 (5, ddd; 10.8, 8.6 and 1.8), 2.51 (6, m), 1.05 (6-Me, d; 7.0), 4.01 (7, dd; 9.6 and 1.9), 4.65 (8, d; 9.6), 7.36-7.41 (10/11/12/13/14, m); leucic acid (D) 4.92 (2, dd; 10.1 and 3.5), 1.76 (3/4, m), 1.45 (3, m), 0.94 (5, d; 6.6), 0.94 (4-Me, d; 6.4); 3-amino-2-methylpropionic acid (C) 2.73(2, m), 1.22 (2-Me, d; 7.2), 3.25 (3, ddd; 13.6, 6.8 and 6.1), 3.54 (3, ddd; 13.5, 6.1 and 3.4), 6.91(3-NH, brt; 6.1); 3-chloro-4-methoxyphenylalanine (B) 4.82 (2, ddd;
8.8, 7.2 and 5.6), 5.64 (2-NH, d; 8.8), 3.03 (3, dd; 15.4 and 7.2), 3.16 (3, dd; 15.4 and 5.6), 7.23 (5, d; 2.2), 3.88 (7-OCH3, s), 6.85 (8, d; 8.5), 7.09 (9, dd; 8.5 and 2.2).
Cryptophvcin 9 To a solution of 10mg of Cryptophycin 1 in 1mL dry methanol was added 10 L
methanolic HCl (obtained by treating 1.25g thionyl chloride with 25mL MeOH).
After stirring for 4 h the solvent was removed in vacuo and the sample was left under vacuum for 12 h. Reversed-phase HPLC gave 8mg of pure Cryptophycin 9.
'H NMR (CDC13): amino or hydroxy acid unit S(carbon position, multiplicity; J
in Hz); 5, 7-dihydroxy-8-methoxy-6-methyl-8 phenyl-2-octenoic acid (A) 5.76 (2, d; 15.5), 6.67 (3, ddd; 15.5, 9.5 and 5.6), 2.34 (4, ddd; 14.1, 11.1 and 9.5), 2.62 (4, dddd; 14.1, 5.6, 1.8 and 1.5), 5.09 (5, ddd; 11.1, 7.8 and 1.8), 2.24 (6, dqd; 7.8, 7.0 and 2.2), 1.03 (6-Me, d; 7.0), 3.71 (7, dd; 8.3 and 2.2), 4.03(8, d; 8.3), 3.20 (8-OCH3, s), 7.31-7.40 (10/11/12/13/14, m); leucic acid (D) 4.86 (2, dd; 9.8 and 3.5), 1.71 (3/4, m), 1.41 (3, m), 0.89 (5/4-Me, d; 6.4); 3-amino-2-methylpropionic acid (C) 2.71 (2, ddq;
6.8, 3.9 and 7.2), 1.21 (2-Me, d; 7.2), 3.23 (3, ddd; 13.5, 6.8 and 6.0), 3.52 (3, ddd;
13.5, 6.0 and 3.9), 6.90 (3-NH, brt; 6.0); 3-chloro-4-methoxyphenylalanine (B) 4.82 (2, ddd; 8.8, 7.4 and 5.7), 5.66 (2-NH, d; 8.8), 3.02 (3, dd; 14.4, 7.4), 3.15 (3, dd; 14.4 and 5.5), 7.23 (5, d; 2.2), 3.87 (7-OCH3, s), 6.84 (8, d; 8.5), 7.08 (9, dd; 8.5 and 2.2).
Cryptoph ci~10 To a stirred solution of 7mg of Cryptophycin 9 in lmL of acetone and 0.3mL
water was added 8 L of 2N NaOH. After stirring for 4 h the solution was neutralized to pH 7 with 1N HCl and the solvent was removed under reduced pressure. The residue was subjected to reversed-phase HPLC using 7:3 MeOH/H20 to yield pure Cryptophycin (5mg).
10 'H NMR (CD3OD): amino or hydroxy acid unit 6(carbon position, multiplicity;
J
in Hz); 5, 7-dihydroxy-8-methoxy-6-methyl-8 phenyl-2-octenoic acid (A) 5.99 (2, dt; 15.4 and 1.3), 6.82 (3, dt; 15.4 and 7.3), 2.30 (4, m), 2.50 (4, m), 3.66 (5, td;
7.8 and 3.5), 2.05 (6, d pentet; 1.8 and 7.0), 0.96 (6-Me, d; 7.0), 4.04 (7, dd; 8.8 and 2.0), 4.01 (8, d; 8.8), 3.12 (8-OCH3, s), 7.26-7.36 (10/11/12/13/14, m); 3-amino-2-methylpropionic acid (C) 2.50 (2, m), 1.02 (2-Me, d; 7.3), 3.16 (3, dd; 13.4 and 6.9), 3.82 (3, dd; 13.4 and 6.6); 3-chloro-4-methoxyphenylalanine (B) 4.57 (2, dd; 8.5 and 6.5), 2.82 (3, dd;
13.9 and 8.6), 3.03 (3, dd; 13.9 and 6.5), 7.25 (5, d; 2.2), 3.82 (7-OCH3, s), 6.96 (8, d;
8.6), 7.13 (9, dd; 8.6 and 2.2). 13C NMR (CD3OD): 8 179.5, 173.4, 168.2, 155.4, 143.7, 141.7, 131.9, 131.7, 129.8, 129.3 (2C), 129.2 (2C), 128.8, 126.2, 123.2, 113.4, 85.9, 74.5, 74.1, 56.8, 56.6, 56.3, 43.3, 41.2, 40.2, 38.8, 38.0, 15.5, 9.9.
Cryptoph cin 12 To a solution of 5mg of Cryptophycins 1, 5 or 8 in lmL of 4:1 acetone/water was added 15 L of 2N NaOH. After stirring at room temperature for 5 h, the reaction mixture was neutralized to pH 7 with 1N HCl and evaporated. The CH2C12-soluble material was passed through a small silica-cartridge with CH2C12, 1:1 EtOAc/CH2C12, and EtOAc. The fraction eluted with EtOAc contained pure Cryptophycin 12.
'H NMR (CD3OD): amino or hydroxy acid unit 6(carbon position, multiplicity; J
- in Hz); 5, 7, 8-trihydroxy-6-methyl-8 phenyl-2-octenoic acid (A) 6.07 (2, ddd; 15.5, 1.3 and 1.2), 6.40 (3, dt; 15.5 and 7.3), 2.49 (4, m), 2.60 (4, m), 3.92 (5, ddd;
9.3, 6.7 and 4.5), 1.94 (6, m), 1.07 (6-Me, d; 6.6), 3.61 (7, dd; 8.9 and 7.6), 4.56 (8, d;
7.6), 7.36 (10/14, dd; 7.4 and 1.5), 7.32 (11/13, brt; 7.5), 7.25 (12, m); 3-amino-2-methylpropionic acid (C) 2.54 (2, ddq; 7.0, 6.6 and 7.0), 1.02 (2-Me, d; 7.0), 3.14 (3, dd;
13.5 and 7.0), 3.42 (3, dd; 13.4 and 6.6); 3-chloro-4-methoxyphenylalanine (B) 4.57 (2, dd;
8.4 and 6.7), 2.83 (3, dd; 13.8 and 8.4), 3.02 (3, dd; 13.8 and 6.6), 7.25 (5, d;
2.1), 3.82 (7-OCH3, s), 6.95 (8, d; 8.5), 7.12 (9, dd; 8.5 and 2.1). Methylation of Cryptophycin 12 with diazomethane gave Cryptophycin 6.
Crvptophvcin 14 To a solution of 3mg of Cryptophycin 6 in 1mL of 3:1 acetone/H20 was added 5 L of 2N NaOH. After stirring for 5 h, the reaction mixture was neutralized to pH 7 with 1N HCl and then evaporated to dryness. The residue was subjected to reversed-phase HPLC to give 2.4mg of Cryptophycin 14.
'H NMR (CD3OD): amino or hydroxy acid unit S(carbon position, multiplicity; J
in Hz); 5-hydroxy-6-methyl-8 phenyl-2, 7-octadienoic acid (A) 5.98 (2, d;
15.3), 6.78 (3, dt; 15.3 and 7.5), 2.35 (4, m), 3.64 (5, td; 7.2 and 4.8), 2.47 (6, m), 1.14 (6-Me, d;
6.9), 6.22 (7, dd; 15.9 and 8.1), 6.39 (8, d, 15.9), 7.24-7.36 (10/11/12/13/14, m); 3-amino-2-methylpropionic acid (C) 2.35 (2, m), 1.02 (2-Me, d; 6.9), 3.18 (3, dd; 13.2 and 6.6), 3.36 (3, dd; 13.2 and 4.5); 3-chloro-4-methoxyphenylalanine (B) 4.58 (2, dd; 8.7 and 6.3), 2.80 (3, dd; 13.8 and 9.0), 3.05 (3, dd; 13.8 and 6.3), 7.25 (5, d;
2.1), 3.82 (7-OCH3, s), 6.95 (8, d; 8.4), 7.13(9, dd; 8.4 and 2.1).
Cryptophycin 35 A catalytic amount of Pt02 was added to a flask containing 0.5mL of CHZC12.
The air in the flask was evacuated, H2 was introduced, and the mixture was stirred at room temperature for 20 min. A solution of 10mg of Cryptophycin 1 in minimum CH2C12 was added and the mixture was stirred at room temperature for 45 min.
The catalyst was removed by filtration through celite/cotton and the solvent was evaporated.
Reversed phase HPLC of the residue on a C18 column yielded 6.5mg of Cryptophycin 35.
EIMS m/z (relative intensity) 656/658 (25/10), 412/414 (25/12), 280/282 (20/10), 195/197 (78/25), 141 (58), 91 (100); high resolution EIMS m/Z 656.2864 (calcd for C35H45C1N20g, 0.0mmu error); 'H NMR (CDC13) amino or hydroxy acid unit S
values (carbon positions, multiplicities; J in Hz) 2,3-dihydro-7,8-epoxy-5-hydroxy-6-methyl-8-phenyl octanoic acid (A) 2.32 (2, ddd; 14.5, 9.2, 5.8), 2.10 (2, ddd; 14.5, 9.2, 6.2), 1.5-1.8 (3/4 overlapping m), 5.07 (5, ddd; 12.5, 5.6, 2.0), 1.80 (6, m), 1.12 (6-Me, d; 7.0), 2.90 (7, dd; 7.4, 1.8), 3.67 (8, d; 1.8), 7.24 (10/14, m), 7.32-7.38 (11/12/13, m); 3-chloro-4-methoxyphenylalanine (B) 4.71 (2, ddd; 8.7, 6.4, 6.3), 5.62 (2-NH, d;
8.7), 3.08 (2H-3, br d; 6.4), 7.19 (5, d; 2.0), 3.87 (7-OMe, s), 6.83 (8, d; 8.5), 7.07 (9, dd;
8.4, 2.0); 3-amino-2-methylpropionic acid (C) 2.72 (2, m), 1.18 (2-Me, d;
6.9), 3.12 (3, ddd; 11.4, 10.6, 5.6), 3.70 (3, ddd), 6.76 (3-NH, br t, 6.0); leucic acid (D) 4.83 (2, dd;
9.9, 3.8), 1.39 (3, m), 1.70 (3, m), 1.72 (4, m), 0.87 (4-Me, d; 5.3), 0.86 (5, d; 5.3);
13C NMR (CDC13) unit 8 values (carbon positions) A 172.4 (1), 36.2 (2), 32.0 (3), 21.1 (4), 76.6 (5), 40.2 (6), 13.6 (6-Me), 63.3 (7), 59.2 (8), 136.8 (9), 125.6 (10/14), 128.7 (11/13), 128.6 (12); B 170.7 (1), 53.7 (2), 35.5 (3), 130.0 (4), 131.1 (5), 122.2 (6), .153.8 (7), 56.1 (7-OMe), 112.1 (8), 128.5 (9); C 175.2 (1), 38.2 (2), 13.6 (2-Me), 42.1 (3); D 171.9 (1), 71.7 (2), 39.6 (3), 24.5 (4), 22.9 (4-Me), 21.4 (5).

Examnie 7 Analysis of Microtubule Depolvmerizing Activity of Cryptophycin Materials Vinbiastine, cytochalasin B, tetramethylrhodarnine isothiocyanate (TRITC)-phalloidin, sulforhodamine B (SRB) and antibodies against 0-tubulin and vimentin were obtained from the Sigma Chemical Company. Basal Medium Eagle containing Earle's salts (BME) was from Gibco and Fetal Bovine Serum (FBS) was purchased from Hyclone Laboratories.
Cell Lines The Jurkat T cell leukemia line and A-10 rat aortic smooth muscle cells were obtained from the American Type Culture Collection and were 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 for resistance to vinblastine (SKVLBI) were a generous gift from Dr. Victor Ling of the Ontario Cancer Institute.
Both cell lines were maintained in BME containing 10% FBS and 501Ag/mL gentamycin sulfate.
Vinblastine was added to a fmal concentration of 1 g/mL to SKVLB1 cells 24 hours after passage to maintain selection pressure for P-glycoprotein-overexpressing cells.
Cell Proliferation and Cycle Arrest Assays Cell proliferation assays were performed as described by Skehan et al.ll For Jurkat cells, cultures were treated with the indicated drugs as described in Skehanll and total cell numbers were determined by counting the cells in a hemacytometer.
The percentage of cells in mitosis was determined by staining with 0.4% Giemsa in PBS

followed by three rapid washes with PBS. At least 1000 cells per treatment were scored for the presence of mitotic figures and the mitotic index was calculated as the ratio of cells with mitotic figures to the total number of cells counted.
Imrnunofluorescence Assays A-10 cells were grown to near-confluency on glass coverslips in BME/10% FBS.
Compounds in PBS were added to the indicated final concentrations and cells were incubated for an additional 24 hours. For the staining of microtubules and intermediate filaments, the cells were fixed with cold methanol and incubated with PBS
containing 10% calf serum to block nonspecific binding sites. Cells were then incubated at 37'C for 60 min with either monoclonal anti-0-tubulin or with monoclonal anti-vimentin at dilutions recommended by the manufacturer. Bound primary antibodies were subsequently visualized by a 45-minute incubation with fluorescein-conjugated rabbit antimouse IgG. The coverslips were mounted on microscope slides and the fluorescence patterns were examined and photographed using a Zeiss Photomicroscope IIl equipped with epifluorescence optics for fluorescein. For staining of microfilaments, cells were fixed with 3% paraformaldehyde, permeabilized with 0.2% Triton X-100 and chemically reduced with sodium borohydride (lmg/mL). PBS containing 100nM TRITC-phalloidin was then added and the mixture was allowed to incubate for 45 min at 37' C.
The cells were washed rapidly three times with PBS before the coverslips were mounted and inunediately photographed as described above.
Effects of cryptonhvcins and vinbiastine on Jurkat cell proliferation and cell cycle Dose-response curves for the effects of cryptophycin compounds and vinblastine on cell proliferation and the percentage of cells in mitosis are indicated in Figures 2A and 2B, respectively. Less than 3% of untreated cells displayed mitotic figures.
Both the cryptophyein-compounds and vinblastine caused dose-dependent increases in the percentage of cells observed in mitosis. The increase in the mitotic index was closely correlated with decreases in cell proliferation, i.e. the concentrations of both cryptophycin compounds and vinblastine that caused 50% of the cells to accumulate in mitosis was virtually the same as the concentration which inhibited cell proliferation by 50%. The IC_4Os for the cryptophycin compounds and vinblastine for these effects were 0.2 and 8nM, respectively.

Effects of cytochalasin B, vinblastine and cryptophycins on the cytoskeleton Aortic smooth muscle (A-10) cells were 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 were visualized by indirect immunofluorescence and microfilaments were stained using TRITC-phalloidin.
The morphological effects of each drug were 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 intermediate filaments. Both vinblastine and the cryptophycin compound caused marked depletion of microtubules. Neither compound affected microfilament organization; however, vimentin intermediate filaments collapsed, forming concentric rings around the nuclei of cells treated with either vinblastine or a cryptophycin compound.
Effects of cryptophycins and vinblastine on taxol-stabilized microtubules A-10 cells were treated for 3 hours with 0 or 10 M taxol before the addition of PBS, l00nM vinblastine or lOnM cryptophycin compound. After 24 hours, microtubule organization was examined by immunofluorescence 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 of microtubules in non-pretreated cells. However, pretreatment with taxol prevented microtubule depolymerization in response to vinblastine.
Similarly, taxol pretreatment completely stabilized microtubules against cryptophycin-induced depolymerization.
Reversibilitv of microtubule depolymerization by vinblastine and cryptophvcin A-10 cells were treated with either lOOnM vinblastine or lOnM cryptophycins for 24 hr, resulting in complete microtubule depolymerization. The cells were then washed and incubated in drug- free medium for periods of 1 hour or 24 hours.
Microtubules repolymerized rapidly after the removal of vinbiastine, showing significant levels of microtubules after 1 hour and complete morphological recovery by 24 hour. In contrast, microtubules did not reappear in cells treated with cryptophycin compounds at either 1 hour or 24 hours after removal of the compound.
Reversibilitv of cryptophycins-. vinblastine- and taxol-inhibition of cell proliferation SKOV3 cells were treated for 24 hours with previously determined IC" doses of vinblastine, cryptophycin compounds or taxol (i.e. values determined in experiments summarized in Table 5). During this time the cell density increased from 0.4 to 0.5 0.05 absorbance units (Figure 3), indicating a 25% increase in cell number for all three treatments. Removal of the drugs resulted in rapid growth of the vinblastine-treated cells, such that their numbers were increased approximately 3-fold in 24 hours. In contrast, cells treated with cryptophycin compounds or taxol remained arrested, increasing only 0.2- to 0.4-fold in the 24 hours following removal of the drug. The proliferative capacity of cryptophycins or taxol-treated cells was subsequently restored since the cells then doubled in the next 24 hours.
Effects of combinations of vinblastine and cryptophycins on cell proliferation SKOV3 cells were treated with combinations of cryptophycins and vinblastine for 48 hours. The percentages of surviving cells were then determined and the IC50s for each combination was calculated. The effects of these combinational treatments, as well as single drug treatments, are depicted as an isobologram (Figure 4). The IC50s for combinations of cryptophycin compounds and vinblastine fell very close to the line of additivity, indicating that these two drugs induce only additive inhibitions of cell proliferation.
Toxicitv of cryptophycins, vinblastine and taxol toward SKOV3 and SKVLB1 cells SKVLB1 cells are resistant to natural product anticancer drugs because of their over expression of P-glycoprotein12. The abilities of taxol, vinbiastine and cryptophycin compounds to inhibit the growth of SKOV3 and SKVLBI cells are summarized in Table 5. Taxol caused dose-dependent inhibition of the proliferation of both cell lines with IC50s for SKOV3 and SKVLB1 cells of 1 and 8000nM, respectively.
Vinblastine also inhibited the growth of both cell lines, with IC50s of 0.35 and 4200nM
for SKOV3 and SKVLB1 cells, respectively. Cryptophycins demonstrated IC50s of 7 and 600pM for SKOV3 and SKVLB1 cells, respectively. The resulting resistance factors for cells to the compounds are calculated as the IC50s for SKVLB1. IC50s for SKOV3 cells are also indicated in Table 5.

Thus it is demonstrated that the present invention provides novel cryptophycin compounds, as well as previously-disclosed cryptophycin compounds, which are potent inhibitors of cell proliferation, acting by disruption of the microtubule network and inhibition of mitosis. The 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 seemed 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 of cryptophycin compounds and vinblastine on the proliferation and cell cycle progression of the Jurkat T-cell leukemia cell line was compared. Both compounds caused parallel dose-dependent inhibitions of cell proliferation and accumulation of cells in mitosis.
Since antimitotic effects are commonly mediated by disruption of microtubules in the mitotic spindles, the effects of cryptophycin compounds on cytoskeletal structures were characterized by fluorescence microscopy. Immunofluorescence staining of cells treated with either a cryptophycin compound or vinblastine clearly demonstrated that both compounds caused the complete loss of microtubules. Similar studies with SKOV3 cells demonstrate that the anti-microtubule effects of cryptophycin compounds are not unique to the smooth muscle cell line. Neither drug affected the levels or distribution of microfilament bundles, as was readily induced by cytochalasin B, indicating that the loss of microtubules may not be due to a non-specific mechanism, e.g. activation of proteases or loss of energy charge. Both vinbiastine and cryptophycin compounds also promote marked collapse of vimentin intermediate filaments, such that brightly staining rings were formed around the cell nucleus.
Removal of vinblastine from the culture medium resulted in rapid repolymerization of microtubules. In contrast, cells treated with cryptophycin compounds remained depleted of microtubules for at least 24 hours after the compound was removed from the cultures.
The present invention demonstrates that cryptophycin compounds circumvent P-glycoprotein-mediated multiple drug resistance. Transport by P-glycoprotein limits the ability of natural product anticancer drugs to inhibit the growth of tumor cells with acquired or de novo drug resistance.13-1s Vinca alkaloids, while very useful in the initial course of chemotherapy, are extremely good substrates for transport by P-glycoprotein, and so are of very limited usefulness against P-glycoprotein-mediated MDR
tumors.
Therefore, identification of agents which overcome multiple drug resistance may, should lead to the development of useful and novel anticancer agents. The cryptophycin compounds of the present invention appear to be such agents since they are poor substrates for P- glycoprotein-mediated transport. This fact is reflected in the low cell resistance factor for cryptophycin compounds compared with vinblastine, taxol and other natural product drugs.
Total Synthesis of Cryptophvcins The structures of the novel synthesized compounds, viz. Cryptophycins 51, 52, 53, 55, 56, 57, 58, and 61 were confirmed in a straightforward manner using methodology that is well-known to those trained in the art. Mass spectral data were consistent with the molecular compositions. Proton and carbon NMR data were very similar to those of cryptophycin 1 and related naturally-occurring and semi-synthetic analogs.
The following examples demonstrate the total synthesis of cryptophycin compounds as well as their use as therapeutic agents in accordance with the invention.
Example 8 Synthesis of Cryptophvcin 51 S-trans-3-Penten-2-ol (A) A mixture of racemic trans-3-penten-2-ol (933mg, 11mmo1), trifluoroethyl laurate (4.14g, 15mmo1), and porcine pancreatic lipase (PPL, 2.0g) in 25mL of anhydrous diethyl ether was stirred for 80 hours. The PPL was then filtered off and washed with ether three times. The ether filtrate was evaporated and the sticky oil was then subjected to short-path vacuum distillation. The S-trans-3-penten-2-ol (A) was condensed in a .
liquid nitrogen cooled trap (383mg). 'H NMR (CDC13) 8 5.57 (4-H; dq, -15.3/6.0), 5.47 (3-H; ddd, -15.3/6.4/1.2), 4.19 (2-H; 1:4:6:4:1 pentuplet, 6.4), 2.24 (OH;
bs), 1.63 (5-H3; d, 6.0), 1.19 (1-H3; d, 6.4). 13C NMR (CDC13) S 135.5 (3), 125.5 (4), 68.7 (2), 23.3 (5), 17.5 (1).

S-trans-2-(2-Prop nloxy)-3 pentene (B) To a vigorously stirred mixture of S-enantiomer A (628mg, 7.3mmol), tetrabutylammonium hydrogen sulfate (138mg, 0.41mmo1), and 40% NaOH in water (5mL) at 0 C was added dropwise propargyl chloride (767mg, 10.3mmol, 7451LL).
Vigorous stirring was continued overnight after which time the mixture was neutralized by HCI at 0 C and the propargyl ether extracted into pentane. The extract was evaporated and the propargyl ether was purified on a short silica gel column (2% diethyl ether/pentane) to give 778mg of propargyl ether B, [a]D -118.9 (c 2.0, CHC13); 'H
NMR (CDC13) 6 5.70 (4-H; dq, 18.5/6.5), 5.31 (3-H; ddd, 18.5/7.2/1.4), 4.15 (1'-H; dd, -15.6/2.1), 4.01 (1'-H; dd, -15.6/2.1), 4.01 (2-H; m), 2.38 (3'-H; t, 2.1), 1.73 (5-H; dd, 6.5/1.4), 1.25 (1H; d, 6.3).
(3R.4R)-4-Methylhept-5(E)-en-l-yn-3-ol (C) An aliquot of butyl lithium hexane solution (2.5M, 5.1mL, 12.8mmol) was evaporated in vacuo and the residue cooled to -90 C. A solution of propargyl ether B
(454mg, 3.66mmol) in lOmL of THF was slowly added. After allowing the temperature to increase to room temperature overnight, the reaction mixture was quenched with NH4C1 solution. Extraction with ether three times, evaporation of the dried extract, and purification of the residue on a silica gel column (5 % EtOAc/hexane) gave 322mg of alcohol C (71% yield), [a]D +32.9 (c 3.0, CHC13); IR (NaCI) v,,,a, 3306, 2968, 1455, 1379, 1029, 975 cm 1. 1H NMR (CDC13) 6 5.61 (6-H; dq, 15.3/6.3), 5.38 (5-H;
dd, 15.3/7.7), 4.13 (3-H; bs), 2.45 (1-H; d, 1.5), 2.38 (4-H; m), 2.20 (OH; bd, 3.3), 1.68 (7-H; d, 6.2), 1.09 (4-CH3; d, 6.8). 13C NMR (CDC13) S 131.5 (5), 127.9 (6), 83.5 (2), 73.6 (1), 66.2 (3), 43.4 (4), 18.1 (7), 15.7 (4-Me).
f3S.4R)-3-tert-Butvldimethylsilvloxy-4-methylhent-5E-enal lD) To a stirred solution of alcohol C (248mg, 2mmol) and imidazole (340mg, 5mmo1) in 3mL of dry DMF was added tert-butyldimethylsilyl chloride (452mg, 3mmo1).
After stirring the mixture overnight, l OmL of 10% NaOH was added to destroy the excess tert-butyldimethylsilyl chloride. The product was extracted into ether and the extract washed successively with water, 0.5 N HCI, and water, dried and evaporated.
Purification of the residue by chromatography on silica gel with hexane gave 457mg of (3R,4R)-3-tert-butyldimethylsilyloxy-4-methylhept-5(E)-en-1-yne (96% yield), 'H NMR
(CDC13) 6 5.50 (6-H; dq, 15.3/6.1), 5.38 (5-H; dd, 15.3/7.5), 4.16 (3-H; dd, 5.7/1.7), 2.37 (1-H; d, 1.7), 2.35 (4-H; m), 1.68 (7-H; d, 6.1), 1.07 (4-Me; d, 6.8), 0.90 (CMe3;
s), 0.12 (SiMe; s), 0.09 (SiMe; s).
Using the same procedure the corresponding TBDPS derivative, (3R,4R)-3-tert-butyldiphenylsilyloxy-4-methylhept-5(E)-en-1-yne, was formed in 92% yield, [a]D +32.9 (c 3.0, CHC13). 'H NMR (CDC13) S 7.72/7.38 (2Ph-H5), 5.32 (6-H; m), 5.25 (5-H;
dd, 16.2/7.3), 4.29 (3-H; dd, 5.2/2.0), 2.38 (4-H; m), 2.33 (1-H; d, 2.0), 1.64 (7-H; d, 5.3), 1.11 (4-Me; d, 6.9), 1.06 (CMe3). 13C NMR (CDC13) 8 136.1/ 135.9 /133.6 /129.7/129.6/127.5/127.3 (Ph), 132.4 (5), 126.1 (6), 83.3 (2), 73.5 (1), 68.0 (3), 43.6 (4), 26.9 (CM%), 19.4 (CMe3), 18.0 (7), 14.7 (4-Me).
2-Methylbutene (1.15mL 2M solution in THF, 2.3mmol) was added to 1.1mL of BH3 THF solution (1M, 1.lmmol) at -25 C and the mixture was stirred in an ice bath for two hours. The temperature was then cooled to -50 C and a solution of the TBS
derivative (238mg, lmmol) in lmL of THF was added all at once. The cooling bath was removed and the reaction mixture was allowed to warm to and remain at room temperature for one hour. Then 2.2 M KH2PO4/K2HPO4 solution (4. 8mL) and 30%

(0.8mL) were added at 0 C. One hour later, the THF was evaporated and the residue was extracted into ether. The dried ether extract was evaporated and the residue chromatographed on silica gel (1 % EtOAc/hexane) to give 194mg of aldehyde D(76%
yield). 'H NMR (CDC13) 8 9.78 (1-H; t, 2.3), 5.46 (6-H; dq, 15.3/6.1), 5.34 (5-H; dd, 15.3/7.5), 4.13 (3-H; m), 2.47 (2-H; m), 2.31 (4-H; m), 1.66 (7-H; br d, 6.1), 0.99 (4-Me; d, 6.8), 0.87 (CMe3; s), 0.07 (SiMe; s), 0.04 (SiMe; s).
The tert-butyldiphenylsilyl ether (TBDPS) derivative of the aldehyde was formed in 83% yield, 1H NMR (CDC13) 6 9.52 (1-H; t, 2.4), 7.69/7.40 (2Ph-HS), 5.28 (6-H; m), 5.22 (5-H; dd, 16.2/6.2), 4.19 (3-H; m), 2.42 (2-H; m), 2.29 (4-H; m), 1.60 (7-H; d, 5.4), 1.07 (CMe3), 1.02 (4-Me; d, 6.9). 13C NMR (CDC13) S 202.0 (1), 136.1/133.6/
133.3 /130.2/129.7/127.7/127.6 (Ph), 132.3 (5), 126.2 (6), 72.8 (3), 47.6 (2), 42.2 (4), 27.1 (CM%), 19.6 (CMe3), 18.3 (7), 14.9 (4-Me).
Methyl (5S.6R)-5-tert-Butyldimethylsilvloxy-6-methyl-7-oxonona-2E.7 E-dienoate (E) To a stirred solution of aldehyde D(0.74g, 2.9mmol) and trimethyl phosphonoacetate (632mg, 3.5mmol) in 5mL of THF cooled to -78 C was added tetramethylguanidine (435 L, 3.5mmo1). After 30 minutes the cooling bath was removed and the mixture was stirred for another four hours. The mixture was neutralized with 1N

HC1 and the product was extracted into ether. Evaporation of the dried ether extract left a residue which was chromatographed on silica gel (5 % EtOAc/hexane) to give 0.814g of E (90% yield). 'H NMR (CDC13) 6 6.93 (3-H;dt, 15.6/7.8), 5.62 (2-H; dd, 15.6/1.2), 5.37 (8-H, m), 5.37 (7-H, m), 3.71 (OCH3, s), 3.61 (5-H, m), 2.29 (4-H2, m), 2.22 (6-H, m), 1.66 (9-H3; br d, 6.1), 0.99 (6-Me; d, 6.8), 0.88 (CMe3; s), 0.03 (SiMe; s), 0.01 (SiMe; s).
The tert-butyldiphenylsilyl ether (TBDPS) derivative of the aldehyde was formed in 90% yield, 'H NMR (CDC13) 6 7.68/7.38 (2Ph-H5), 6.75 (3-H;dt, 15.6/7.4), 5.62 (2-H; d, 15.6), 5.34 (8-H, m), 5.29 (7-H, m), 3.70 (5-H, m), 3.68 (OCH3, s), 2.28 (4-H2, m), 2.20 (6-H, m), 1.62 (9-H3; d, 5.3), 1.08 (CMe3), 0.99 (6-Me; d, 6.9). 13C
NMR
(CDC13) S 166.7 (1), 146.4 (3), 136.0/134.2/133.8/129.62/129.56/127.5/127.4 (Ph), 132.5 (7), 125.8 (8), 122.6 (2), 76.2 (5), 51.3 (OCH3), 41.7 (6), 36.8 (4), 27.0 (CM~e ), 19.4 (CMe3), 18.1 (9), 14.7 (6-Me).
Methvl (5S.6R)-5-tert-Butyldimethylsilyloxv-6-methvl-7-oxohept-2(E)-enoate (F) Ozone was passed through a solution of methyl ester E (328mg, 1.0mmo1) and 971LL of pyridine in 15mL of CH2C12 at -78 C and the progress of the ozonolysis was monitored by TLC analysis. After the methyl ester had been consumed, about 500mg of zinc dust and lmL of glacial acetic acid were added. The temperature was slowly increased to 25 C. The mixture was filtered and the filtrate was washed successively with saturated CuSO4 and NaHCO3 solutions. After evaporation of the solvent, the crude aldehyde F (249mg, 83 %) was used in the next step without further purification. 'H
NMR (CDC13) 8 9.96 (7-H; t, 2.3), 6.96 (3-H; dt, 15.7/7.6), 5.90 (2-H; dd, 15.7/0.7), 4.05 (5-H; m), 3.74 (OMe; s), 2.51 (6-H; m), 2.45 (4-H2; m), 1.09 (6-Me; d, 6.9), 0.88 (CMe3; s), 0.04 (SiMe; s), 0.03 (SiMe; s).
Methyl (5S.6R)-5-t-butvldimethvlsilvloxv-6-methvl-8-phenyl-octa-2E 7 E-dienoate (G) To a stirred solution of aldehyde F(25.(kmg, 0.08mmo1) in 1.5mL of THF at -78 C was added 0.80mL of a cold (-78 C) mixture of benzyltriphenylphosphonium chloride (268mg, 0.69mmol, in 6.9mL of THF) and n-butyl lithium (280 L, 2.5M
in hexane). After 15 min, the cold bath was removed and stirring was continued for 2 h.
The reaction was quenched with saturated ammonium chloride solution and the THF was evaporated. The concentrate was extracted with hexane twice and the combined extract was washed with brine, dried and evaporated. The residual oil, a 5:1 mixture of the E

and Z isomers, was dissolved in 1.5mL of benzene containing thiophenol (0.02M) and 1,1'azobis(cyclohexanecarbonitrile) (VAZO, 0.006M) and the mixture was refluxed for 5h. After cooling to RT, hexane (15mL) was added and the organic solution was washed successively with 10% NaOH and brine, dried (MgSO4), and evaporated.
Chromatography of the residue on silica gel (2% EtOAc/hexane) led to 24mg (80%) of G, [a]D +68.2 (c 1.5, CHC13); EIMS mlz 374 (< 1%; M+), 359 (1; M+-CH3), 317 (10;
M+-Bu), 275 (10), 243, (73), 143 (20), 115 (10), 97 (64), 89 (31), 73 (100);
HREIMS
m/z 374.2232 (C=H34O3Si, 0+4.5mmu), 359.2031 (C21H31O3Si, 0+1.1mmu), 317.1579 (C18HuO3Si, A-0.6mmu); UV (MeOH) a,1187C (e) 206 (33500), 252 (20100) nm; IR
P.
2952, 2855, 1725, 1657, 1435, 1257, 1168, 1097, 970, 836, 775 cm-1; 'H NMR 8 7.2-7.4 (Ph-H5; m), 6.96 (3-H; ddd, 15.6/7.8/7.5), 6.37 (8-H; d, 15.9), 6.16 (7-H;
dd, 15.9/8.1), 5.84 (2-H; d, 15.6), 3.75 (5-H; ddd, 10.2/6.0/4.2), 3.72 (OMe; s), 2.44 (6-H;
m), 2.36 (4-H2; m), 1.10 (6-Me; d, 6.9), 0.91 (Si-CMe3; s), 0.06 (Si-Me; s), 0.05 (Si-Me; s); 13C NMR S 166.8 (1), 146.4 (3), 137.6 (Ph 1'), 131.9 (8), 130.4 (7), 128.5 (Ph 3'/5'), 127.0 (Ph 4'), 126.0 (Ph 2'/6'), 122.9 (2), 75.0 (5), 51.4 (OMe), 42.8 (6), 37.6 (4), 25.9 (Si-CMe--J, 18.1 (Si-CMe3), 16.2 (6-Me), -4.4 (Si-Me), -4.5 (Si-Me). Calcd for C=H34O3Si: C, 70.52; H, 9.17. Found: C, 70.72; H, 9.42.
(5S,6R)-5-t-Butvldimethylsilyloxy-6-methyl-8-phenylocta-2E.7 E-dienoic acid (H) To a solution of ester G (159mg, 0.43mmo1) in 7mL acetone was added 5mL of 1N aq LiOH. The mixture was stirred at 25 C for 3h, diluted with 20mL of Et20, and acidified to .= pH 4 with iN HC1. The organic layer was separated and washed with 20mL portions of brine and water, dried (MgSO4) and evaporated. Chromatography of the residual oil on silica gel with 40% EtOAc in hexane containing 0.5% AcOH
resulted in pure acid H as a pale yellow mobile oil (145mg, 95% yield): [a]D +87.0 (c 1.4, CHC13); EIMS m/z; 343 (1; M+-OH), 303 (5), 275 (9), 257 (4), 229 (62), 213 (16), 171 (22), 143 (37), 131 (16), 115 (23), 97 (100), 91 (44); HREIMS m/z 343.2107 (CZ1H31O2Si, A-1.3mmu), 229.1220 (C15H1702, 0+0.9mmu); UV X. (e) 206 (24500), 252 (15600) nm; IR P. 3300-2800 (br), 2956, 2856, 1697, 1651, 1419, 1256, 1097, 836, 693 cm 1; 'H NMR 8 10.4 (CO2H; bs, W1,2 =100), 7.2-7.4 (Ph-H5; m), 7.09 (3-H;
ddd, 15.6/7.6/7.6), 6.39 (8-H; d, 15.9), 6.16 (7-H; dd, 15.9/8.1), 5.85 (2-H;
d, 15.6), 3.78 (5-H; ddd, 6.0/6.0/4.2), 2.46 (6-H; m), 2.40 (4-H2; m), 1.12 (6-Me; d, 6.9), 0.92 (Si-CMe3; s), 0.07 (SiMe2; s); 13C NMR d 171.6 (1), 149.1 (3), 137.5 (Ph 1'), 131.8 (8), 130.5 (7), 128.5 (Ph 3'/5'), 127.1 (Ph 4'), 126.1 (Ph 2'/6'), 122.7 (2), 74.9 (5), 42.9 (6), 37.6 (4), 25.8 (Si-CM~e ), 18.1 (Si-CMe3), 16.1 (6-Me), -4.4 (Si-Me), -4.5 (Si-Me).
2.2.2-Trichloroethyl Ester of 3-(3-Chloro-4-methoxyphenyl)-D-alanine (I) A sample of the D-chlorotyrosine BOC derivative (160mg, 0.35mmol) was dissolved in 3mL neat trifluoroacetic acid and allowed to stand at room temperature for lh. Removal of the excess reagent under reduced pressure returned the desired amine I
,as the trifluoroacetate salt (165mg, 100% yield), [ ]D +1.7 (c 5.2, CHC13);
IR v1i87C
3400-2500 (br), 1760, 1680, 1500, 1200, 1130, 1070, 805, 710 cm 1; 1H NMR S
8.07 (NH2; br m, W1/2 =45), 7.27 (5-H; s), 7.12 (9-H; d, 8.1), 6.88 (8-H; d, 8.1), 4.86/4.67 (CH2CC13; AB q, -12.0), 4.41 (2-H; bs, W l/2 = 20), 3.86 (OMe; s), 3.33 (3-H;
dd, -14.4/3.6), 3.22 (3-H'; dd, -14.4/6.6); 13C NMR 8 167.6 (1), 155.0 (7), 130.9 (5), 128.8 (9), 125.4 (4), 123.1 (6), 112.7 (8), 93.4 (CC13), 75.3 (CH2CC13), 56.1 (OMe), 54.2 (2), 34.9 (3).
Compound J
To a stirred solution of H (25mg, 0.07mmo1) in 3mL of anhydrous DMF under argon was added successively pentafluorodiphenylphosphinate (FDPP, 32mg, 0.08mmo1), trifluoroacetate salt I (35mg, 0.07mmo1) and diisopropylethylamine (DIEA, 27mg, = 36 L, 0.21mmo1, = 3 equiv). Stirring was continued at 25 C for lh and then the reaction mixture was extracted with 20mL of EtZO. The ether extract was washed with lOmL of 1N HCI, followed by lOmL of sat'd NaHCO3, 2OmL of brine and 20mL of water, dried (MgSO4), and evaporated. The residual pale yellow oil was subjected to chromatography on silica gel (15% EtOAc in hexane) to give J as a colorless oil (32mg, 65% yield): [a]D +11.8 (c 1.2, CHC13); EIMS m/z; 644/646/648/650 (7/8/6/3; M+-Bu), 570/572/574 (46/100/21), 536/538 (18/15), 394/396 (67/29), 275 (20), 155/157 (29/9);
HREIMS m/z 644.0981 (C29H34C14NOSSi, A-2.13mmu); UV N'. (e) 204 (54900), 230 (23200), 248 (19200), 284 (3500) mn; IR vn= 3290, 2980, 2850, 1760, 1680, 1640, 1505, 1380, 1270, 1169, 990, 720 cm-'; 1H NMR unit A 8 7.2-7.4 (Ph-H5; m), 6.87 (3-H; ddd, 15.0/7.8/7.5), 6.37 (8-H; d, 16.2), 6.18 (7-H; dd, 16.2/8.1), 5.82 (2-H; d, 15.0), 3.75 (5-H; ddd, 9.9/6.0/4.8), 2.46 (6-H; m), 2.36 (4-H2; m), 1.11 (6-Me; d, 6.9), 0.91 (SiCMe3; s), 0.07 (SiMe; s), 0.06 (SiMe; s); unit B S 7.19 (5-H; d, 2.1), 7.04 (9-H;
dd, 8.4/2.1), 6.85 (8-H; d, 8.4), 5.85 (NH; d, 7.8), 5.08 (2-H; ddd, 7.8/6.0/5.7), 4.81/4.74 (CH2CC13; AB q, -11.7), 3.87 (OMe; s), 3.22 (3-H; dd, -14.1/5.7), 3.12 (3-H'; dd, -14.1/6.0). 13C NMR unit A 6 165.1 (1), 143.0 (3), 137.6 (9), 132.0 (8), 130.4 (7), 128.5 (11/13), 127.0 (12), 126.0 (10/14), 124.7 (2), 75.0 (5), 42.6 (6), 37.6 (4), 25.9 (Si-CMe3), 18.1 (Si-CMe3), 16.5 (6-Me), -4.3 (Si-Me), -4.6 (Si-Me); unit B 6 170.1 (1), 154.3 (7), 131.1 (5), 128.5 (4/9), 122.6 (6), 112.2 (8), 94.2 (CC13), 74.8 (CHZCCI3), 56.1 (OMe), 53.0 (2), 36.5 (3).
(1'R.5S.6R)-N-1'-(carbo-2".2".2"-trichloroethoxy)-2'-(3-chloro-4-met hoxyphen 1=~yl-5=
t-butvldimethylsilyloxv-6-methyl=8-phenyl-octa-2E .7E-dienamide (K) To a solution of J (50mg, 0.07mmo1) in 4mL MeCN was added 400 L of 50% aq HF and the mixture stirred for 1h at 25 C. Extraction into 30mL of EtZO, followed by washing the ether extract with 30mL portions of sat'd NaHCO3, brine and water, drying (MgSO4) and evaporation, gave alcohol K as a colorless foam (40mg, 95% yield):
[a]D -6.1 (c 1.3, CHC13); EIMS m/z (rel intensity) 587/589/591/593 (M+, < 1%), 552/554/556 (1/2/0.5), 456/458/460/462 (1/2/1/0.2), 342/344/346 (7/8/4), (15/5), 195/197 (6/2), 155/157 (99/34), 131 (100), 91 (77); HREIMS m/z 587.0721 (C27H29 5C14NO5, 0+7.9mmu); UV )4. 204 (56500), 230 (22100), 248 (18100), 284 (3600) nm; IR v,,m 3400, 3300, 2980, 1780, 1680, 1640, 1505, 1270, 1180, 1090, 1000, 770 cm-'. 'H NMR unit A 8 7.2-7.4 (Ph-H5; m), 6.92 (3-H; ddd, 15.3/7.8/7.5), 6.46 (8-H; d, 15.9), 6.14 (7-H; dd, 15.9/8.4), 5.90 (2-H; d, 15.3), 3.65 (5-H; ddd 7.8/5.6/4.0), 2.39 (6-H/4-H2; bm), 1.78 (OH; bs, W1n=40 Hz), 1.14 (6-Me; d, 6.9); unit B 6 7.18 (5-H; d, 1.8), 7.03 (9-H; dd, 8.4/1.8), 6.84 (8-H; d, 8.4), 5.97 (NH; d, 7.8), 5.06 (2-H;
ddd, 7.8/6.0/5.7), 4.79/4.72 (CH2CC13; AB q, -12.0), 3.86 (OMe; s), 3.20 (3-H;
dd, -14.1/5.7), 3.10 (3-H'; dd, -14.1/6.0). 13C NMR unit A 6 165.3 (1), 142.6 (3), 137.0 (9), 131.7 (8), 131.0 (7), 128.5 (11/13), 127.3 (12), 126.1 (10/14), 125.0 (2), 73.8 (5), 43.2 (6), 37.2 (4), 16.8 (6-Me); unit B 6 170.2 (1), 154.2 (7), 131.0 (5), 128.4 (9), 128.3 (4), 122.5 (6), 112.2 (8), 94.2 (CC13), 74.7 (CH2CC13), 56.1 (OMe), 53.0 (2), 36.5 (3).
3-(tert-Butoxxcarbonyl)amino-2.2-dimethylpropanoic Acid (M) To a solution of 3-amino-2,2-dimethylpropan-l-ol (L) (3.0g, 29mmo1) in 51mL of a 10 % solution of triethylamine in MeOH was added di-tert-butyl dicarbonate (6.7g, 31mmo1) and the mixture was stirred at 25 C for lh. After removal of solvent, the residue was dissolved in CH2C12 (30mL) and the solution was washed twice with KHSO4 (pH 2) and once with saturated NaCI solution, and dried (MgSO4). Removal of solvent in vacuo afforded 5.8g (93 % yield) of 3-(tert-butoxycarbonyl)amino-2,2-dimethylpropan-l-ol as a white solid which was directly used for the next step without further purification (> 95 % pure by NMR analysis), mp 70.5-71.5 C; IR P.
3350, 1685, 1456 cm'1; 'H NMR 8 4.87 (NH; br s), 3.72 (OH; br s), 3.19 (1-H2; d, 5.1), 2.95 (3-H2; d, 6.0), 1.44 (CMe3; s), 0.85 (2-Me2; s); 13C NMR (CDC13) 6 157.6 (BOC
CO), 79.7 (CMe3), 68.1 (1), 47.1 (3), 36.7 (2), 28.3 (CMe3), 22.4 (2-Me2).
To a solution of alcohol 3-(tert-butoxycarbonyl)amino-2,2-dimethylpropan-l-ol (5.3g, 25.9mmo1) and sodium periodate (16.6g, 77.7mmo1) in carbon tetrachloride (52mL), acetonitrile (52mL) and water (78mL) was added ruthenium trichloride hydrate (122mg), and the mixture was stirred at 25 C for lh. The mixture was filtered through Celite and a saturated solution of potassium carbonate in water (50mL) was added. The water layer was separated, washed with ether (20mL), acidified with HCl to pH
2 at 0 C
and extracted with CH2C12 (30mL x 3). The combined extracts were washed with saturated NaCI solution and dried (MgSO4). Removal of solvent in vacuo yielded a residue that was first subjected to flash reversed-phase chromatography on a C18 silica (ODS 120A, 50 to 90% MeOH) and then crystallized from ether to give 3.7g (66%
yield) of M as a white solid, mp 106-108 C; EIMS m/z (rel intensity) 217 (0.1), 161 (11), 98 (25), 88 (71), 57 (100); HREIMS m/z 217.1292 (C,oH19N04, 0+2.2mmu); IR P. 3450-2500, 1710, 1694, 1510 cm 1; 'H NMR of major conformer S 5.03 (NH; br s), 3.26 (3-H2; m), 1.45 (CMe3; s), 1.24 (2-Me2; s); 13C NMR (CDC13) a 183.2 (1), 156.3 (BOC
CO), 79.6 (CMe3), 49.5/47.9 (2/3), 28.4 (CM~e ), 22.9 (2-Me2).
Allyl (2S)-2-Hydroxv-4-methvlpentanoate (N) To a solution of 2.66g of L-leucic acid (20mmo1) and 1.74g of sodium bicarbonate (20mmo1) in 30mL water at 0 C was added 30mL of a CH2C12 solution of 6.44g of tetrabutylammonium chloride (20mmo1) and 1.74mL of allyl bromide (20mmo1).
After vigorously stirring the mixture for 24h, the CHZC12 was evaporated. About 50mL
water was added and the aqueous layer was extracted four times with EtZO. The ether solution was dried over anhydrous sodium sulfate and then evaporated. The residue was passed ---- through a short Si column to give 3.21g of allyl ester N (93% yield) as a colorless oil, [a]D -8.4 (c 1.1, CHC13); IR P. 3464, 2957, 1732, 1203, 1140, 1087 cm 1; 1H

5.92 (allyl 2-H; m), 5.34 (allyl 3-Hz; dd, 17.4/1.1), 5.28 (allyl 3-HE, dd, 10.5/1.1), 4.67 (allyl 1-H2; d, 5.7), 4.23 (2-H; br s), 2.64 (OH; br s), 1.89 (4-H; m), 1.57 (3-H2; m), 0.96 (5-H3; d, 6.5), 0.95 (4-Me; d, 6.7); 13C NMR 6 175.3 (1), 131.4 (allyl C-2), 118.6 (3), 68.9 (2), 65.7 (allyl C-1), 43.2 (3), 24.1 (4), 23.0 (5), 21.3 (4-Me).
Allyl (2S)-2-(3'(tert-Butoxvcarbonyl)amino-2' 2'-dimeth lnropanoylox1 -4-methvlpentanoate (0) To a solution of 0.8g of M(3.7mmo1), 0.76g of N(4.4mmol), and 92mg DMAP
in lOmL of dry CHZC12 at 0 C was added 0.84g of DCC (4.lmmol) in CH2C12. The mixture was stirred at 25 C for 3h and filtered. The filtrate was washed with saturated aqueous sodium bicarbonate, dried (Na2SO4), and evaporated in vacuo. Flash chromatography (silica gel, 5% EtOAc/hexane) afforded 1.0g (92% yield) of pure 0 as a colorless oil, Rf 0.68 (17:83 EtOAc/hexane), [a] D-29.4 (c 18.1, CHC13); EIMS
m/z (rel intensity) 371 (2, M+), 242 (13), 184 (12), 126 (20), 84 (100); HREIMS
m/z 371.2317 (C19H33N06, A-0.9mmu); IR (neat) v,,.3385, 2963, 1731, 1720, 1513 cm 1; 1H
NMR (300 MHz, CDC13) unit C 8 5.39 (NH; obscured br s), 3.33 (3-H; dd, -13.5/7.4), 3.27 (3-H'; dd, -13.5/5.9), 2.78 (2-H, m), 1.44 (CMe3; s), 1.23 (2-Me; s), 1.22 (2-Me;
s); unit D 8 5.91 (allyl 2-H; ddt, 16.6/10.3/6.0 Hz), 5.34 (allyl 3-Hz; bd, 16.6), 5.27 (allyl 3-HE; bd, 10.3), 5.08 (2-H; dd, 9.6/3.6), 4.65 (allyl 1-H2; m), 1.6-1.9 (3-HZ/4-H;
m), 0.94 (5-H3; d, 6.3), 0.94 (4-Me; d, 7.3). 13C NMR unit C 8 176.5 (1), 156.3 (BOC
CO), 79.0 (CMe3), 48.6 (3), 44.0 (2), 28.4 (CMe=), 22.2/23.0 (2-Me2); unit D 8 170.6 (1), 131.4 (allyl C-2), 119.1 (allyl C-3), 70.9 (2), 66.0 (allyl C-1), 39.5 (3), 24.8 (4), 23.0 (5), 21.5 (4-Me).
(2S)-2-[3'(tert-Butoxvcarbonvl)amino-2' 2'-dimethylpropanovloxyl-4-methvlpentanoic Acid To lOmL of a solution of 180mg (0.49mmol) of 0 and 60mg (0.05mmol) of tetrakis(triphenylphosphine)palladium in dry THF (under argon atmosphere) was slowly added 470 L (5.4mmol) of dry morpholine over 10 min. After stirring for 50 min, 40mL of ether was added and the solution was washed with 1N HCl (4OmL) and then extracted with saturated sodium bicarbonate (2 x 30mL). The aqueous extract was acidified with 0.5N HCl and extracted with ether (40mL). The ether extract was washed with water (2 x 30mL), dried (MgSO4) and evaporated in vacuo to give P as a colorless mobile oil (152mg, 95%); [a]D -22.2 (c 2.2, CHC13); EIMS m/z (rel intensity) 331 (1, M+), 275 (1), 258 (4), 231 (9), 202 (36), 174 (13), 144 (31), 126 (16), 114 (14), 98 (54), 88 (50), 84 9100); HREIMS m/z 331.2004 (C16H29N06, 0-1.Ommu). 'H NMR

(CDC13) unit C 6 5.41 (NH; dd, 5.7/5.4), 3.30 (3-H2; m), 2.68 (2-H; m), 1.43 (CMe3; br s), 1.22 (2-Me; s), 1.21 (2-Me; s); unit D S 6.47 (1-OH; br s, W1/2 = 35), 5.09 (2-H; dd, 9.3/2.7), 1.7-1.9 (3-H2/4-H; m), 0.97 (5-H3; d, 6.0), 0.94 (4-Me; d, 6.0). 13C
NMR
(CDC13) unit C 8 176.5 (1), 156.5 (BOC CO), 79.3 (CMe3), 48.6 (3), 44.0 (2), 28.3 (CMe ), 23.0 (2-Me), 22.2 (2-Me); unit D S 175.4 (1), 70.6 (2), 39.5 (3), 24.8 (4), 23.0 (5), 21.4 (4-Me).
Compound 0 To a solution of alcohol K (80mg, 0.14mmo1), acid P (68mg, 0.21mmo1) and DMAP (4mg) in dry CH2C12 (4mL) stirred at 0 C under an argon atmosphere was added DCC (44mg, 0.21mmo1) in dry CH2C12 (1mL). The mixture was stirred at 0'C for minutes, during which time a white precipitate developed, and then allowed to warm to room temperature and stirred for a further 4 hours. The precipitate was filtered off and the filtrate diluted with Et10 (4OmL) and washed successively with dilute HCl (1M, 40mL), saturated NaHCO3 (4OmL) and brine (4OmL). The ethereal layer was dried (MgSO4) and evaporated in vacuo to give a waxy solid. Chromatography (silica, EtOAc:hexane, 1:3) led to pure Q as a colorless, viscous oil (103mg, 84%), [a]D -3.1 (c 2.9, CHC13); EIMS m/z 800/802/804/806 (<1, M+-C5H8O2), 415/417/419/421 (5/3/3/2), 342/344/346 (7/9/4), 286/288/290 (2/6/2), 207 (34), 178 (22), 155/157 (66/24), 131 (36), 91 (70), 70 (100); HRRiMS m/z 800.2179 (C38H4$N20835C14, 0-1.4mmu); UV (MeOH) X. (e) 204 (51200), 230 (18500), 248 (17200), 282 (2200) nm; IR (NaCI) vjõ.
3376, 2965, 1755, 1728, 1712, 1678, 1504, 1258, 1150, 1067, 732 cm'. iH NMR (CDC13) unit A: 7.28-7.33 (10-H/14-H/11-H/13-H; m), 7.22 (12-H; m), 6.78 (3-H; ddd, 15.8/6.4/6.3), 6.40 (8-H; d, 15.8), 6.01 (7-H; dd, 15.8/8.7), 5.88 (2-H; d, 15.8), 5.06 (5-H; bm, WõZ = 20 Hz), 2.62 (6-H; m), 2.53 (4-H2; bm, WI,2 = 15 Hz), 1.12 (6-CH3; d, 6.8); unit B 7.18 (5-H; d, 2.0), 7.05 (9-H; dd, 8.5/2.0), 6.83 (8-H; d, 8.5), 6.49 (NH; d, 7.9), 5.06 (2-H; bm, Wla = 20 Hz), 4.79/4.70 (CH2CC13; AB q, -11.7), 3.85 (OCH3; s), 3.20 (3-Hb; dd, -14.1/5.8), 3.07 (3-H,; dd, -14.1/6.7); unit C 5.38 (NH; bt, 6.5), 3.27 (3-H2; d, 6.5), 1.20 (2-CH3; s), 1.15 (2-CH3'; s); unit D 4.92 (2-H; dd, 10.0/3.8), 1.72 (4-H; bm, Wõ2=20 Hz), 1.67 (3-Hb; ddd, -14.1/10.0/5.0/), 1.56 (3-Ha; ddd, -14.1/9.1/3.8), 1.43 (CO2CMe3; s), 0.86 (4-CH3; d, 6.4), 0.82 (5-H3; d, 6.4).

(CDC13) S unit A 165.4 (1), 139.3 (3), 136.9 (9), 131.7 (8), 130.1 (7), 128.6 (11/13), 127.5 (12), 126.2 (10/14), 125.4 (2), 76.5 (5), 41.1 (6), 33.4 (4), 16.7 (6-Me); unit B

170.0 (1), 154.1 (7), 131.2 (5), 128.8 (4), 128.5 (9), 122.3 (6), 112.1 (8), 94.3 (CH CC13), 74.6 (CH2CC13), 56.1 (7-OMe), 53.2 (2), 36.6 (3); unit C 176.9 (1), 156.4 (CO2CMe3), 79.1 (CO CMe3), 48.7 (3), 44.0 (2), 22.8 (2-Me), 22.3 (2-Me'); unit D
170.7 (1), 71.4 (2), 39.5 (3), 28.4 (CO2CMe=), 24.8 (4), 23.0 (4-Me), 21.4 (5).
Amino Acid R
To the amino acid Q (100mg, 0.11mmol) was added activated Zn dust (400mg, excess) and AcOH (4mL). The heterogenous mixture was subject to sonication for minutes, stirred for a further 90 minutes at room temperature, and then poured onto a pad of Celite. The organic material was washed from the Celite pad with CH2C12.
The solvent was removed in vacuo, leaving the carboxylic acid as a colorless amorphous solid.
Without purification the crude acid was dissolved in trifluoroacetic acid (TFA, 5mL) and allowed to sit at room temperature for 1 hour. After this time excess TFA was removed in vacuo and the resulting amorphous solid was then subjected to chromatographic purification (Sep-PakT", silica, initially CHZC12 then 10%
MeOH/CH2C12), yielding the trifluoroacetate ammonium salt of the desired compound.
Repeated lyophilization of an aqueous solution of the salt resulted in the free amino acid R as a colorless amorphous solid (68mg, 91 % over two steps); IR (NaC1) P.
3300, 3200, 2965, 1693, 1606, 1504, 1441, 1259, 1201, 1146, 1066, 727 cm''. 'H NMR
(CD3OD) 8 unit A: 7.33 (10-H/14-H; d, 7.4), 7.28 (11-H/13-H; t, 7.4), 7.18-7.23 (12-H;
m), 6.69 (3-H; ddd, 15.6/7.7/7.0), 6.43 (8-H; d, 15.8), 6.04 (7-H; dd, 15.8/8.9), 6.00 (2-H; d, 15.6), 5.01 (5-H; ddd, 9.1/6.9/3.1), 2.64 (4-Hb; bm, Wlt2 _= 30 Hz), 2.60 (6-H;
bm, WIn = 20 Hz), 2.49 (4-H8; ddd, 15.8/9.1/7.7), 1.13 (6-Me; d, 6.7); unit B
7.18-7.23 (5-H; m), 7.11 (9-H; dd, 8.3/1.6), 6.92 (8-H; d, 8.3), 4.59 (2-H; bm, W1t2 =20 Hz), 3.81 (OCH3; s), 3.14 (3-Hb; dd, -13.7/4.3), 2.96 (3-H$; m, W1,2 = 20 Hz); unit C 2.96 (3-H2; bm, W1/2.= 20 Hz), 1.31 (2-CH3; s), 1.25 (2-CH3'; s); unit D 4.90 (2-H;
dd, 9.6/4.0), 1.66 (4-H; bm, Wl/2= 25 Hz), 1.59 (3-Hb; ddd, -14.4/9.6/4.8), 1.53 (3-Ha; ddd, -14.4/9.1/4.0), 0.81 (4-Me; d, 6.5), 0.74 (5-H3; d, 6.5). 13C NMR (CD3OD) 6 unit A
167.7 (1), 140.7 (3), 138.4 (9), 133.0 (8), 131.7 (7), 129.6 (11/13), 128.5 (12), 127.3 (10/14), 127.1 (2), 78.4 (5), 43.1 (6), 35.7 (4), 17.4 (6-Me); unit B 179.8 (1), 155.2 (7), 132.3 (4), 132.1 (5), 130.1 (9), 123.0 (6), 113.4 (8), 56.6 (7-OMe), 56.6 (2), 37.8 (3), unit C 176.8 (1), 48.2 (3), 42.2 (2), 23.3 (2-Me), 23.3 (2-Me'); unit D 172.0 (1), 73.4 (2), 40.7 (3), 26.0 (4), 23.1 (4-Me), 21.8 (5).

Cryptophvcin 51 To a stirred solution of amino acid T (75mg, 0.11mmo1) in anhydrous DMF
(2OmL) at room temperature under argon was added diisopropylethylamine (DIEA, 44mg, 60 L, 0.34mmol, --3 equiv.) followed by pentafluorodiphenylphosphinate (FDPP, 55mg, 0.14mmo1, --1.3 equiv.) in DMF (2mL). The mixture was stirred for 12 hours, Et20 (4OmL) was added, and the ether layer was washed successively with HCI (1M, 40mL), brine (4OmL) and H20 (40mL), dried (MgSO4) and evaporated under reduced pressure.
The residual waxy solid was further purified by reverse phase chromatography (ODS, , 30% H20/MeCN, 3mL miri') to give Cryptophycin 51 as a colorless aznorphous 10 solid (45mg, 61 %), [a]D +26.4 (c 0.25, CHC13); EIMS m/z 652/654 (M+, 3/1), 632/634 (3/2), 426/428 (51/15), 227 (64), 195/197 (64/22), 155/157 (71/15), 131 (59), 91 (100); HREIMS m/z 652.2936 (C36H45N20735C1, A-2.1mmu); UV (MeOH) A. (e) 204 (52000), 228 (20400), 250 (13400), 284 (2800) nm; IR (NaCI) P. 3376, 3270, 2960, 1747, 1721, 1659, 1536, 1514, 1259, 1150, 1066, 1013, 980, 694 cm''. 'H NMR
(CDC13) 8 unit A 7.32 (10-H/14-H; dd, 8.0/1.5), 7.29 (11-H/13-H; t, 8.0), 7.24 (12-H;
bm, WõZ = 15 Hz), 6.77 (3-H; ddd, 15.2/10.8/4.3), 6.40 (8-H; d, 15.8), 6.01 (7-H; dd, 15.8/8.8), 5.76 (2-H; dd, 15.2/1.1), 5.04 (5-H; ddd, 11.1/6.4/1.9), 2.54 (4-Hb/6-H; bm, W1,2 = 15 Hz), 2.37 (4-Ha; ddd, -14.3/11.1/10.8 ), 1.13 (6-Me; d, 6.8); unit B
7.20 (5-H;
d, 2.0), 7.05 (9-H; dd, 8.4/2.0), 6.84 (8-H; d, 8.4), 5.61 (NH; d, 7.8), 4.74 (2-H; ddd, 7.8/7.6/5.4), 3.87 (OMe; s), 3.11 (3-Hb; dd, -14.2/5.4), 3.06 (3-H$; dd, -14.2/7.6); unit C 7.24 (NH; bm, W12 =15 Hz), 3.40 (3-Hb; dd, -13.5/8.5), 3.12 (3-H8; dd, -13.5/3.6), 1.22 (2-Me; s), 1.15 (2-Me', s); unit D 4.85 (2-H; dd, 10.2/3.6), 1.66 (3-Hb;
ddd, -14.0/10.2/4.6), 1.61 (4-H; bm Wl,2= 20.0 Hz), 1.33 (3-H8; ddd, -14.0/9.0/3.6), 0.74 (4-Me; d, 6.6), 0.72 (5-H3; d, 6.6). 13C NMR (CDC13) 6 unit A 165.1 (1), 142.2 (3), 136.7 (9), 131.7 (8), 130.1 (7), 128.6 (11/13), 127.5 (12), 126.1 (10/14), 124.6 (2), 77.0 (5), 42.2 (6), 36.5 (4), 17.3 (6-Me); unit B 170.3 (1), 154.1 (7), 130.9 (5), 129.5 (4), 128.3 (9), 122.5 (6), 112.3 (8), 56.1 (7-OMe), 54.2 (2), 35.3 (3); unit C 178.0 (1), 46.5 (3), 42.7 (2), 22.8 (2-Me), 22.6 (2-Me'); unit D 170.6 (1), 71.5 (2), 39.5 (3), 24.5 (4), 22.7 (4-Me), 21.2 (5).

Example 9 Synthesis of Cryptophvcin 52 and Cryptophycin 53 To a stirred solution of Cryptophycin 51 (75mg, 0.12mmol) in anhydrous dichloromethane (7.5mL) at 0 C under argon was added a solution of m-chloroperbenzoic acid (mCPBA, 50mg, 0.23mmol, = 2 equiv. based on 80% active oxygen) in dichloromethane (1mL). After 30 minutes the reaction mixture was allowed to warm to room temperature and stirred for a further 12 hours. The solvent was then removed under reduced pressure to give a 1.8:1 mixture of Cryptophycins 52 and 53 (by NMR
analysis), respectively, as an amorphous solid. The mixture of regioisomeric epoxides was dissolved in minimal acetonitrile and subjected to reverse phase chromatography (YMC-ODS, 10 , 250mm x 22.5mm, 30% H20/MeCN, 6mL min') to separate Cryptophycin 52 (37mg, 48 %) and Cryptophycin 53 (19mg, 25 %).
Snectral data for Cryptophvcin 52 ja]D +19.9 (c 0.5, CHC13); EIMS mlz 668/670 (4/2, M+), 445 (35), 244 (12), 227 (22), 195/197 (66/27), 184 (45), 155/157 (38/10), 91 (100); HREIMS m/z 668.2873 (C36H45N2 0 835C1, 0-0.9mmu), 445.2497 (C25H35NO6, A-3.3mmu); UV (MeOH) X. (e) 204 (35100), 218 (20900) nm; IR (NaCI) v. 3415, 3270, 2960, 1748, 1721, 1650, 1536, 1504, 1260, 1192, 1150, 1066, 1013, 800, 698 cm 1. 1H NMR (CDC13) 6 unit A
7.33-7.38 (11-H/12-H/13-H; bm, W1/2=25 Hz), 7.24 (10-H/14-H; m, W1n=15 Hz), 6.76 (3-H; ddd, 15.1/10.8/4.3), 5.71 (2-H; dd, 15.1/1.7), 5.20 (5-H; ddd, 11.0/5.0/1.8), 3.68 (8-H; d, 1.9), 2.92 (7-H; dd, 7.5/1.9), 2.57 (4-Hb; ddd, -14.6/1.8/1.7), 2.45 (4-Ha;
ddd, -14.6/11.0/10.8), 1.78 (6-H; bm, W12=15 Hz), 1.14 (6-Me; d, 6.9); unit B
7.18 (5-H; d, 2.2), 7.04 (9-H; dd, 8.4/2.2), 6.83 (8-H; d, 8.4), 5.56 (NH; d, 7.9), 4.73 (2-H;
ddd, 7.9/7.4/5.3), 3.87 (OMe; s), 3.09 (3-Hb; dd, -14.6/5.3), 3.05 (3-Ha; dd, -14.6/7.4);
unit C 7.20 (NH; dd, 8.6/3.2), 3.41 (3-Hb; dd, -13.4/8.6), 3.10 (3-He; dd, -13.4/3.2), 1.22 (2-Me; s), 1.15 (2-Me'; s); unit D 4.82 (2-H; dd, 10.2/3.5), 1.73 (3-Hb;
bm, W1,2 = 20 Hz), 1.66 (4-H; bm, W1,2 = 20 Hz), 1.31 (3-H8; ddd, -13.8/9.1/3.5), 0.84 (4-Me; d, 6.6), 0.82 (5-H3; d, 6.6); 13C NMR (CDC13) S unit A 164.9 (1), 141.8 (3), 136.7 (9), 128.7 (11/13), 128.3 (12), 125.6 (10/14), 124.7 (2), 75.9 (5), 63.0 (7), 59.0 (8), 40.7 (6), 36.9 (4), 13.5 (6-Me), unit B 170.3 (1), 154.1 (7), 130.9 (5), 129.5 (4), 128.5 (9), 122.6 (6), 112.4 (8), 56.1 (7-OMe), 54.3 (2), 35.3 (3), unit C 178.0 (1), 46.5 (3), 42.8 (2), 22.8 (2-Me), 22.8 (2-Me'), unit D 170.5 (1), 71.2 (2), 39.3 (3), 24.6 (4), 22.7 (4-Me), 21.2 (5).

Spectral data for Cryptophycin 53 (a]D +20.8 (c 1.7, CHC13); EIMS m/z 668/670 (5/4, M ), 445 (32), 244 (15), 227 (24), 195/197 (64/21), 184 (60), 155/157 (33/9), 91 (100); HREIMS m/z 668.2853 (C36H45N20 8 35C1, Al.lmmu); UV (MeOH) X11187C (e) 204 (38700), 218 (22900) nm; IR
(NaCI) v. 3415, 3280, 2917, 2849, 1748, 1722, 1660, 1504, 1465, 1260, 1190, 1150, 1066, 755 cm'. 'H NMR (CDC13) 6 unit A 7.29-7.36 (11-H/12-H/13-H, bm, W112=20 Hz), 7.23 (10-H/14-H; dd, 8.3/1.7), 6.77 (3-H; ddd, 15.1/10.9/4.3), 5.81 (2-H;
dd, 15.1/1.3), 5.17 (5-H; ddd, 11.2/4.9/1.8), 3.58 (8-H; d, 1.7), 2.90 (7-H; dd, 7.8/1.7), 2.67 (4-Hb; ddd, 14.7/11.2/10.9), 2.56 (4-Ha; dddd, 14.7/4.3/1.8/1.3), 1.67-1.78 (6-H;
bm, W1,2.= 45), 1.03 (6-CH3; d, 7.1); unit B 7.21 (5-H; d, 2.1), 7.07 (9-H;
dd, 8.5/2.1), 6.84 (8-H; d, 8.4), 5.90 (2-NH; d, 7.9), 4.75 (2-H; ddd, 7.9/7.9/4.9), 3.85 (7-OCH3; s), 3.14 (3-Hb; dd, 14.5/4.9), 3.03 (3-Ha; dd, 14.5/7.9); unit C 7.29-7.36 (3-NH;
bm, W122 = 25), 3.43 (3-Hb; dd, 13.7/8.8), 3.10 (3-He; dd, 13.7/3.4), 1.23 (2-CH3;
s), 1.17 (2-CH3'; s); unit D 4.92 (2-H; dd, 10.3/3.2), 1.67-1.78 (3-Hb/4-H; bm, Wõ2 =
45), 1.48 (3-Ha; ddd, 13.9/8.8/3.2), 0.89 (4-CH3; d, 6.6), 0.86 (5-H3; d, 6.6). 13C NMR
(CDC13) 6 unit A 165.1 (1), 142.0 (3), 137.0 (9), 128.5 (11/13), 128.5 (12), 125.3 (10/14), 124.6 (2), 76.7 (5), 63.2 (7), 56.2 (8), 40.8 (6), 36.7 (4), 13.4 (6-Me); unit B
170.4 (1), 154.0 (7), 130.8 (5), 129.7 (4), 128.2 (9), 122.5 (6), 112.3 (8), 56.1 (7-OMe), 54.4 (2), 35.3 (3); unit C 177.9 (1), 46.4 (3), 42.7 (2), 23.0 (2-Me), 22.7 (2-Me'); unit D
170.5 (1), 71.3 (2), 39.2 (3), 24.7 (4), 22.8 (4-Me), 21.3 (5).
Example 10 Synthesis of Cryptophycin 55 To a solution of Cryptophycin 52 (6mg, 0.009mmol) in 0.6mL of 2:1 1,2-dimethoxyethane/water was added 2 L of 12 N HCI. The solution was allowed to stir at room temperature for 20 h, neutralized with potassium carbonate, filtered through a 5 filter, and evaporated. The acetonitrile-soluble material was purified by reversed-phase HPLC on C18 (250 x 10mm column) using 4:1 MeOH/H20 to obtain 3.0mg of Cryptophycin 55 (48%). [a]D +42.5 (c 1.1, CHC13); EIMS m/z 704/706/708 (M+ <
1), 668/670 (1.5/0.5, M+-HCl), 445 (6), 226 (8), 195/197 (16/5), 184 (10), 155/157 (33/11), 135 (100), 91 (99), 77 (30); HREIMS m/z 668.2873 (M+-HCI, C36H45N20835C1, A-0.8mmu); UV (MeOH) A. (e) 204 (48400), 218 (29200), 284 (1600) nm; IR (NaCI) v,,,.
3410, 3286, 2959, 1748, 1723, 1666, 1538, 1504, 1455, 1257, 1178, 1066, 753 cm-'.

1H NMR (CDC13) 6 unit A 7.35-7.42 (10-H/11-H/12-H/13-H/14-H; m), 6.78 (3-H;
ddd, 15.1/10.6/4.5), 5.78 (2-H; dd, 15.1/1.7), 5.16 (5-H; ddd, 11.1/8.3/2.1), 4.65 (8-H; d, 9.7), 4.01 (7-H; bd, 9.7), 2.69 (4-Hb; dddd, -14.5/4.5/2.1/1.7), 2.50 (6-H;
bm, Wln.= 15), 2.38 (4-He; ddd, -14.5/11.1/10.6), 1.53 (7-OH, s), 1.04 (6-Me, d, 7.1); unit B 7.21 (5-H; d, 2.2), 7.07 (9-H; dd, 8.5/2.2), 6.85 (8-H; d, 8.5), 5.57 (2-NH;
d, 7.8), 4.74 (2-H; ddd, 7.8/7.6/5.2), 3.88 (7-OCH3; s), 3.13 (3-Hb; dd, 14.5/5.2), 3.05 (3-H8;
dd, 14.5/7.6); unit C 7.21 (3-NH; m), 3.38 (3-Hb; dd, 13.5/8.3), 3.17 (3-Hg;
dd, 13.5/4.1), 1.23 (2-CH3; s), 1.17 (2-CH3'; s), unit D 4.93 (2-H; dd, 10.1/3.5), 1.78 (3-Hb; ddd, 13.5/10.1/5.0), 1.72 (4-H; bm, W1n=20), 1.43 (3-Ha; ddd, 13.5/8.8/3.5), 0.92 (4-CH3; d, 6.6), 0.92 (5-H3, d, 6.4). 13C NMR (CDC13) b unit A 165.1 (C-1), 142.4 (C-3), 138.4 (C-9), 129.0 (C-i1/13), 128.3 (C-12), 128.0 (C-10/14), 124.6 (C-2), 76.1 (C-5), 74.1 (C-7), 62.0 (C-8), 38.4 (C-6), 36.5 (C-4), 8.6 (6-Me); unit B 170.3 (C-1), 154.1 (C-7), 130.9 (C-5), 129.6 (C-4), 129.2 (C-9), 122.6 (C-6), 112.3 (C-8), 56.1 (7-OMe), 54.3 (C-2), 35.3 (C-3); unit C 177.8 (C-1), 46.5 (C-3), 42.8 (C-2), 22.9 (2-Me), 23.0 (C-2-Me'); unit D 170.3 (C-1), 71.3 (C-2), 39.7 (C-3), 24.8 (C-4), 22.7 (4-Me), 21.6 (C-5). The corresponding diol, Cryptophycin 56 (2.8mg, 44% yield), was also obtained.
Example 11 Synthesis of Cryptophycin 57 A small amount of Pt02 (=1mg) was added to a flask containing 0.5mL of CH2C12. The air in the flask was evacuated, H2 was introduced, and the mixture was stirred at room temperature for 20 minutes. A solution containing 10.2mg of Cryptophycin 52 (0.015mmo1) in CHZCIZ (0.3mL) was added and the mixture stirred at room temperature for a further 30 minutes. The catalyst was removed by filtration through Celite/cotton and the solvent was removed in vacuo. Reverse phase HPLC
of the residue (ODS, 101t, 250 x 22.5mm, MeCN/H20 (3:1), 5mL min') yielded pure Cryptophycin 57 (9.1mg, 89%). [a]D +3.4 (c=4.5, CHC13); EIMS m/z 670/672 (M+, 9/3), 447 (10), 246 (63), 229 (20), 195/197 (78/25), 184 (58), 155/157 (39/13), 128 (21), - 91 (100), 77 (23); HREIMS m/z 670.3037 (C36H47N2O835C1, A-1.6mmu); UV (MeOH) X. (e) 204 (31400), 218 (12000), 284 (1200) nm; 'H NMR (CDC13) S unit A: 7.30-7.37 (11/12/13-H, bm), 7.23 (10/14-H, bdd, 7.9, 1.9), 5.03 (5-H, ddd, 9.0, 5.6, 3.4), 3.66 (8-H, d, 2.1), 2.89 (7-H, dd, 7.7, 2.1), 2.27 (2-Hb, ddd, 14.3, 8.7, 6.2), 2.04 (2-Ha, ddd, 14.3, 8.8, 6.8), 1.64-1.75 (6-H/4-H2, bm), 1.61 (3-H2, bm, W,n = 25), 1.11 (6-CH3, d, 7.1), unit B: 7.19 (5-H, d, 2.1), 7.04 (9-H, dd, 8.3, 2.1), 6.83 (8-H, d, 8.3), 5.55 (2-NH, d, 8.3), 4.65 (2-H, ddd, 8.3, 7.3, 5.3), 3.87 (7-OCH3, s), 3.16 (3-Hb, dd, 14.3, 7.3), 3.08 (3-He, dd, 14.3, 5.3), unit C. 6.91 (3-NH, dd, 6.4, 6.4), 3.41 (3-Hb, dd, 13.5, 6.4), 3.30 (3-Ha, dd, 13.5, 6.4), 1.21 (2-CH3, s), 1.13 (2-CH3', s), unit D:
4.80 (2-H, dd, 9.8, 4.1), 1.64-1.75 (3-Hb/4-H, bm), 1.34 (3-H8, ddd, 15.4, 10.1, 4.1), 0.86 (4-CH31 d, 6.5), 0.84 (5-H3, d, 6.5); 13C NMR (CDC13) 8 unit A: 172.6 (1), 136.9 (9), 128.7 (11/13), 128.5 (12), 125.6 (10/14), 76.8 (5), 63.4 (7), 59.2 (8), 40.2 (6), 36.2 (2), 32.2 (4), 21.4 (3), 13.6 (6-Me), unit B: 170.4 (1), 154.0 (7), 131.1 (5), 130.0 (4), 128.5 (9), 122.5 (6), 112.2 (8), 56.1 (7-OMe), 54.3 (2), 35.3 (3), unit C.- 177.6 (1), 47.0 (3), 43.1 (2), 22.5 (2-Me'), 22.4 (2-Me), unit D: 171.7 (1), 72.0 (2), 39.0 (3), 24.6 (4), 22.8 (4-Me), 21.8 (5).

Example 12 Synthesis of Cryptophycin 58 To a stirred solution of Cryptophycin 57 (5.5mg, 0.008mmo1) in 3mL of ethanol free chloroform at =-60 C was added TMSC1 (Used as obtained from Aldrich, =
4.5mg, ,= 5.2 L, = 0.04mmo1). The reaction mixture was stirred for 20 minutes, by which time tlc indicated no starting material remained. The volatile components were then removed under reduced pressure to leave an amorphous solid. This material was taken up in acetonitrile and subject to HPLC (ODS, 10 , 250 x 22.5mm, MeCN/H20 (3:1), 5mL
min') to return pure Cryptophycin 58 (5.4mg, 93%) as the major product. [a]D
+7.2 (c=2.1, CHC13); EIMS m/z 706/708/710 (M+, 27/23/8), 670/672 (M+-HCI, 14/13), (54), 581 (53), 485 (23), 483 (21), 447 (34), 294 (21), 282 (39), 246 (57), (87/27), 184 (73), 155/157 (45/10), 128 (30), 91 (95), 77 (30), 69 (100);
HREIMS mlz 706.2844 (C36H48N20835C12, 0-5.6mmu), m/z 670.3070 (M+-HCI, C36H47N20835C1, A-4.9mmu); UV (MeOH) X. (e) 204 (331900), 218 (11800), 284 (1800) nm; 'H NMR
(CDC13) 6 unit A: 7.34-7.42 (10/11/12/13/14-H, bm), 5.01 (5-H, ddd, 9.6, 8.3, 2.5), 4.65 (8-H, d, 9.6), 4.00 (7-H, dd, 9.6, 1.9), 2.42 (6-H, ddq, 8.3, 1.9, 7.0), 2.29 (2-Hb, ddd, 14.3, 9.4, 4.5), 2.06 (2-H., ddd, 14.3, 8.3, 7.5), 1.62-1.82 (3-H2/4-H2, bm), 0.99 (6-CH3, d, 7.0), unit B: 7.20 (5-H, d, 2.1), 7.06 (9-H, dd, 8.3, 2.1), 6.84 (8-H, d, 8.3), 5.62 (2-NH, d, 8.3), 4.61 (2-H, ddd, 8.3, 7.7, 5.4), 3.87 (7-OCH31 s), 3.17 (3-Hb, dd, 14.3, 7.7), 3.11 (3-H., dd, 14.3, 5.4), unit C: 6.97 (3-NH, dd, 6.4, 6.2), 3.43 (3-Hb, dd, 13.4, 6.2), 3.31 (3-Ha, dd, 13.4, 6.4), 1.23 (2-CH3, s), 1.16 (2-CH3', s), unit D: 4.93 (2-H, dd, 10.0, 4.0), 1.86 (3-Hb, ddd, 14.0, 10.0, 5.5), 1.58 (3-He, ddd, 14.0, 8.3, 4.0), 0.97 (4-CH3, d, 6.8), 0.94 (5-H3, d, 6.6); 13C NMR (CDC13) 6 unit A: 172.8 (1), 138.7 (9), 129.0 (12), 128.9 (11/13), 128.0 (10/14), 76.5 (5), 73.8 (7), 62.1 (8), 38.1 (6), 35.9 (2), 31.8 (4), 21.4 (3), 8.7 (6-Me), unit B: 170.6 (1), 153.9 (7), 131.0 (5), 130.2 (4), 128.5 (9), 122.4 (6), 112.2 (8), 56.1 (7-OMe), 54.4 (2), 35.0 (3), unit C:
177.2 (1), 47.0 (3), 43.2 (2), 22.5 (2-Me'), 22.4 (2-Me), unit D: 171.8 (1), 72.0 (2), 39.4 (3), 24.9 (4), 22.9 (4-Me), 21.7 (5).

Example 13 Synthesis of Cryptophvcin 61 To a solution of Cryptophycin 53 (5mg, 0.007mmol) in 0.5mL of dry benzene was added triphenylphosphine sulfide (4mg, 0.014mmo1) followed by 0.65 L of trifluoroacetic acid as a solution in dry benzene (100 L). The solution was allowed to stir at room temperature for 6 h, neutralized with sodium bicarbonate, filtered and evaporated. The residue was partitioned between water and CH2C12. The CH2C12-soluble material was purified by reversed-phase HPLC on C18 using 4:1 MeCN/H20 to obtain pure Cryptophycin 61 (1.9mg, 37 %). [a]D +28.4 (c=0.7, CHC13); EIMS m/z 684/686 (M+, not observed), 652/654 (M+-S, 5/4), 426/428 (90/29), 294 (10), 227 (100), (57/20), 184 (20), 155/157 (34/9), 131 (45), 129 (44), 91 (76), 77 (27);
HREIMS m/z 652.2973 (M+-S, C3J'45N20 735C1, 0-5.8mmu); UV (MeOH) X. (e) 204 (26700), 218 (11600), 284 (820) nm; IR (NaCI) v. 3410, 3271, 2958, 1749, 1724, 1670, 1503, 1463, 1258, 1176, 1066, 758 cm'. 'H NMR (CDC13) 6 unit A 7.29-7.34 (11/12/13-H; m), 7.25 (10/14-H, bd, 6.6), 6.73 (3-H, ddd, 15.2/10.6/4.5), 5.66 (2-H; dd, 15.2/1.7), 5.22 (5-H, ddd, 11.2/4.2/2.0), 3.68 (8-H, d, 5.1), 3.01 (7-H, dd, 8.4/5.1), 2.52 (4-Hb, dddd, -14.4/4.5/2.0/1.7), 2.41 (4-Ha, ddd, -14.4/11.2/10.6), 1.68-1.74 (6-H, m), 1.14 (6-Me, d, 6.9); unit B 7.18 (5-H, d, 2.2), 7.04 (9-H, dd, 8.4/2.2), 6.84 (8-H, d, 8.4), 5.45 (NH, d, 7.8), 4.75 (2-H, ddd, 7.8/7.3/5.4), 3.87 (OMe, s), 3.09 (3-Hb, dd, -14.5/5.4), 3.05 (3-Hõ dd, -14.5/7.3); unit C 7.17 (NH, dd, 8.3, 3.9), 3.39 (3-Hb, dd, -13.5/8.3), 3.14 (3-H., dd, -13.5/3.9), 1.23 (2-Me, s), 1.16 (2-Me', s); unit D 4.86 (2-H, dd, 10.2/3.4), 1.77 (3-Hb, ddd, -14.0/10.2/4.9), 1.68-1.74 (4-H, m), 1.42 (3-H., ddd, -14.0/8.7/3.4), 0.92 (4-Me, d, 6.6), 0.88 (5-H3, d, 6.4). 13C NMR (CDC13) 6 unit A 164.9 (1), 141.7 (3), 138.3 (9), 128.8 (11/13), 128.0 (12), 126.7 (10/14), 124.7 (2), 76.6 (5), 45.8 (7), 43.9 (8), 43.9 (6), 36.6 (4), 16.0 (6-Me), unit B 170.2 (1), 154.1 (7), 130.9 (5), 129.4 (4), 128.3 (9), 122.8 (6), 112.4 (8), 56.1 (7-OMe), 54.2 (2), 35.3 (3), unit C
177.9 (1), 46.5 (3), 42.7 (2), 22.9 (2-Me), 22.8 (2-Me'), unit D 170.4 (1), 71.3 (2), 39.4 (3), 24.7 (4), 22.7 (4-Me), 21.4 (5).

Example 14 Synthesis of Cryptophvcin 81 Compound S
Compound S is the tert-butyidiphenylsilyl ether (TBDMS) derivative of F.
Compound T
To lOmL of a THF solution of p-methoxybenzyltriphenylphosponium chloride (lmmol) at -78 C was added 4001AL of butyl lithium solution (lmmol, 2.5M in hexane).
The mixture was stirred for 30 minutes and then a 2.64mL aliquot was added to 3mL of a THF solution of aldehyde S(75.Omg, 0.24mmol) at -78 C. After 30 minutes, cooling was ceased, but stirring was continued for another two hours during which time the temperature rose slowly to 25 C. The reaction was quenched with saturated ammonium chloride solution and the THF was evaporated. The products were extracted with hexane twice and the combined organic layer was washed with brine, dried and then concentrated. The residue was applied to a flash silica column (3 %
EtOAc/hexane) to give 63mg of compound T and 40mg of a mixture of T and the Z isomer.
Compound T had the following properties: [a]D+110.5' (CHC13, c 0.75); IRv.
2956, 2857, 1724, 1608, 1511, 1428, 1250, 1173, 1111, 1037, 821, 703, 505 cnm-1;
EIMS m/Z (relative intensity %) 497 (< 1, M+-OMe), 471 (31, M+-Bu), 367 (56), (31), 199 (75), 135 (100); high-resolution EIMS 497.24770 (calcd for C32H37O3Si, A+3.5mmu, M+-OMe), 471.19859 (calcd for C29H31O4Si, A+0.6mmu, M+-Bu'). 'H
NMR 8 7.71/7.68 (SiPh2, 2'-H, 6'-H/2"-H, 6"-H; d; 6.5), 7.45/7.43 (SiPh2, 4'-H/4"-H;
t; 7.4), 7.39/7.38 (SiPh2, 3'-H, 5'-H/3"-H, 5"-H; dd; 7.4, 6.5), 7.24 (10-H, 14-H; d;
8.7), 6.85 (11-H, 13-H; d; 8.7), 6.79 (3-H; dt; 15.7, 7.5), 6.19 (8-H, d, 16.1), 6.00 (7-H, dd, 16.1, 8.1), 5.66 (2-H, dt, 15.7, 1.3), 3.82 (5-H, m), 3.81 (-OCH3, S), 3.69 (CO2CH3, S), 2.41 (6-H, m), 2.36 (4-H, m), 2.30 (4-H, m), 1.12 (6-CH3, d, 7.0), 1.09 (CMe3, S). 13C NMR 8 166.7 (1), 158.8 (12), 146.0 (3), 136.0 (SiPh2, 2', 6'/2", 6"), 134.1/133.7 (SiPh2, 1'/1"), 130.4 (9), 130.0 (8), 129.7/129.6 (SiPh2, 4'/4"), 129.5 (7), 127.6/127.5 (3', 5'/ 3' , 5"), 127.1 (10, 14), 122.8 (2), 113.9 (11, 13), 76.4 (5), 55.2 (OCH3), 51.3 (CO2CH3), 42.1 (6), 37.1 (4), 27.0 (CMe3), 19.5 (CMe3), 16.2 (6-CH3).

Additional compound T was obtained from the mixture of T and the Z-isomer.
The 40mg of the mixture of E and Z isomers was dissolved in 4mL of a benzene solution containing thiophenol (0.02M) and ACN (0.006M). The mixture was heated under reflux for 5h. Work-up and purification by a short Si column give 37.2mg compound T.
Compound U
To 6mL acetone solution of compound T (76mg, 0.15mmol) was added 4.4mL of iN LiOH in water. The clear solution was stirred overnight. Acetone was evaporated and the aqueous solution was acidified with 1N HCI. The product was extracted with EtOAc three times. The organic layer was dried and concentrated. Purification by silica colunm (20% EtOAc/hexane with 0.5 % AcOH) gave 62.2mg acid of compound U (81 %);
[a]D +120.8 (CHC13, c 3.1); IR P. 2960, 2858, 1695, 1650, 1511, 1427, 1250, 1111, 1036, 702 cm 1; 'H NMR 8 7.73/7.70 (SiPh2, 2'-H, 6'-H/2"-H, 6"-H, d, 7.0), 7.50 (SiPh2, 4'-H/4"-H, m), 7.44 (SiPh2, 3'-H, 5'-H/ 3"-H, 5"-H, m), 7.29 (10-H, 14-H, d, 8.6), 6.96 (3-H; dt; 15.6, 7.8), 6.89 (11-H, 13-H, d, 8.6), 6.22 (8-H, d, 16.0), 6.03 (7-H, dd, 16.0, 7.9), 5.70(2-H, d, 15.6), 3.88 (5-H, m), 3.83 (OCH3, S), 2.43 (6-H, m), 2.40 (4-H, m), 1.17 (6-CH3, d, 6.9), 1.14 (CMe3, s); 13C NMR, 171.7 (1), 158.8 (12), 148.8 (3), 135.0 (SiPh2, 2', 6'/2", 6"), 133.9/133.7 (SiPh2, 1'/1"), 130.3 (9), 130.0 (8), 129.7 (SiPh2, 4'/4"), 129.4 (7), 127.6 (SiPh2, 3', 5'/3", 5"), 127.1 (10, 14), 122.5 (2), 113.9 (11, 13), 76.2 (5), 55.2 (OCH3), 42.3 (6), 37.1 (4), 27.0 (CMe=), 19.5 (CMe3), 16.0 (6-CH3).
Compound V
Compound U (59mg, 0.12mmol), the trifluoroacetate salt of compound I (57.2mg, 0.12mmol) and diisopropylethylamine (DIEA, 62 L, 0.36mmol) were dissolved in 1.5mL
of dry DMF. To this solution was added FDPP (55mg, 0.14mmo1), in 0.6mL DMF) and the reaction mixture was stirred for two hours. Ether was added and the organic layer was washed successively with iN HCI, saturated sodium bicarbonate, and brine, respectively. Concentration and purification by chromatography (silica column, 8%
EtOAc/hexane) gave 74.2mg of compound V (72%); [a]D +53.8 (CHC13, c 1.6); IR
P.
3286, 2959, 1760, 1667, 1640, 1607, 1510, 1253, 1174, 1111, 1067, 1027, 703 cm'';
EIMS m/z (relative intensity %) 798/799/800/801/802/803/804/805 (31/14/44/17/23/10/6/3, M+-Bu), 766 (40), 694/695/696/697/698/699/700/701 (70/31/100/38/58/19/14/5), 662 (67), 622 (71), 544 (70), 518 (83); high-resolution EIMS

798.1443 (calcd for C4oH40C14NO6Si, A-6.4mmu, M+-Bu'). 'H NMR 6 unit A
7.69/7.65 (SiPh2, 2'-H, 6'-H/2"-H, 6"-H; d; 6.5), 7.41 (SiPh2, 4'-H/4"-H, m), 7.35 (SiPh2, 3'-H;
5'-H/3"-H, 5"-H, m), 7.24 (10-H, 14-H; d, 8.7), 6.85 (11-H, 13-H, d, 8.7), 6.65 (3-H, .
dt, 15.3, 7.5), 6.20 (8-H, d, 16.1), 6.03 (7-H, dd, 16.1, 8.0), 5.50(2-H, d, 15.3), 3.81 (OCH3, S), 3.77 (5-H, m), 2.39 (6-H, m), 2.34 (4-H, m), 2.29 (4-H', m), 1.11 (6-Me, d, 6.9), 1.06 (CMe3, S); unit B 7.15 (5-H, d, 1.8), 7.00 (9-H, dd, 8.4, 1.8), 6.83 (8-H, d, 8.4), 5.65 (NH, d, 7.7), 5.01 (2-H, ddd, 7.7, 6.0, 5.5), 4.78/4.72 (CH2CC13, ABq, -11.9), 3.86 (OMe, S), 3.15 (3-H; dd, 6.1, -14.5), 3.08 (3-H', dd, 5.8, -14.5).

S unit A 165.1 (1), 158.8 (12), 142.5 (3), 136.0 (SiPh2, 2', 6'/2", 6"), 134.2/133.6 (SiPh2, i'/1"), 130.4 (9), 129.9 (8), 129.7/129.6 (SiPh2, 4'/4"), 129.5 (7), 127.6/127.5 (SiPh2, 3', 5'/3", 5"), 127.1 (10, 14), 124.6 (2), 113.9 (11, 13), 76.4 (5), 55.2 (OMe), 42.1 (6), 37.2 (4), 27.0 (CM~e. ), 19.5 (CMe3), 16.6 (6-Me); unit B 170.0 (1), 154.2 (7), 131.0 (5), 128.4 (4/9), 122.5 (6), 112.1 (8), 94.2 (CC13), 74.7 (CH2CC13), 56.1 (OMe), 52.9 (2) 36.4 (3).
Compound W
To a solution of compound V (55.8mg, 0.065mmo1) in 5.7mL of acetonitrile was added 2.OmL of 49% hydrofluoric acid at 0 C. The ice bath was removed after five minutes and the reaction mixture was stirred vigorously for 17 hours. The product was extracted into ether and the extract was washed successively with saturated sodium bicarbonate and brine. Concentration and normal-phase chromatography (silica column, 25% EtOAc/hexane) gave 31.6mg of compound W(79%); [a]D -3.7 (CHC13, c 1.3);
IR
3286, 2961, 1756, 1668, 1634, 1607, 1510, 1251, 1175, 1066, 812 cm'. 1H NMR S
unit A 7.27 (10-H, 14-H, d, 8.5), 6.93 (3-H, dt, 15.4, 7.6), 6.83 (11-H, 13-H, d, 8.5), 6.34 (8-H, d, 15.9), 5.88 (7-H, dd 15.9, 8.2), 5.86 (2-H, d, 15.4), 3.81 (OMe, S), 3.80 (5-H, m), 2.40 (6-H, m), 2.36 (4-H, m), 1.13 (6-Me, d, 6.8); unit B 7.17 (5-H, d, 1.9), 7.05 (9-H, dd, 8.5, 1.9), 6.83 (8-H, d, 8.5), 5.90 (NH, d, 7.7), 5.03 (2-H, ddd, 7.8, 5.9, 5.6), 4.79/4.72 (CH2CC13, ABq, -11.9), 3.86 (OCH31 S), 3.20 (3-H, dd, 6.0, -14.3), 3.09 (3-H', dd, 5.9, -14.3). 13C NMR S unit A 165.2 (1), 159.1 (12), 142.6 (3), 131.3 (9), 129.8 (7), 128.7 (8), 127.3 (10, 14), 125.0 (2), 114.0 (11, 13), 73.8 (5), 55.3 (OMe), 43.3 (6), 37.2 (4), 16.9 (6-Me) unit B 170.1 (1), 154.2 (7), 131.0 (5), 128.5 (4/9), 122.5 (6), 112.2 (8), 94.2 (CC13), 74.7 (CH2CC13), 56.1 (OMe), 53.0 (2), 36.5 (3).

(2S.2'R)-2-f3'(tert-Butoxycarbonyl)amino-2'-methvlpropanoyl-oxyl-4-m ethylnentanoic acid (AC) A solution of methyl (S)-(+)-3-hydroxy-2-methylpropanoate (X) (lOg, 85mmol) in 300mL of ca. 9M ammonia in methanol was heated to 50 C in a sealed glass flask for 168h, flushed with nitrogen to remove excess ammonia, and then evaporated to dryness in vacuo. The residue was triturated with ether, leaving behind (S)-3-hydroxy-2-methylpropanamide (5.7g, 66% yield) as a white solid, mp 85 . 5-87.5 C; [a]
D+28. 7 (c 3.5, MeOH); EIMS m/z (rel intensity) 88 (19, M-Me), 85 (35), 73 (69); HREIMS
m/z 88.0397 (C3H6NO2, 0+0.2mmu); IR vtõ. 3384, 2960, 1671, 1473 crri 1; 1H NMR 6 5.83 (NH; br s), 5.42 (NH; br s), 3.73 (3-H2; m), 2.55 (2-H; m), 1.19 (2-Me; d, 7.2); 13C
NMR S 180.7 (1), 65.4 (3), 44.0 (2), 14.5 (2-Me). Anal. Found: C, 46.45; H, 8.83.
Calcd for C4H9NO2: C, 46.59; H, 8.79.
A suspension of (S)-3-hydroxy-2-methylpropanamide (2.1g, 20mmo1) in anhydrous THF (2OmL) was added slowly to 1M borane-THF complex (61mmo1, 61mL) cooled to 0 C. The mixture was refluxed for 6h, cooled to 0 C, carefully decomposed with conc HC1 (lOmL), and concentrated in vacuo. The concentrate was saturated with NaOH
(20g), extracted with CHC13 (15mL x 4), and the combined extracts were dried (MgSO4).
After filtration and removal of solvent, distillation in vacuo yielded 1.4g (77% yield) of (R)-3-amino-2-methylpropan-l-ol (Y) as a colorless oil, bp 110-112 C (40mmHg);
[a]D
+8.9 (c 22.6, MeOH); IR v. 3358, 1873, 1598, 1466 cm 1; 'H NMR S 5.18 (NH2;
br s), 3.8 (1-H2; m), 2.95 (3-H; m), 2.68 (3-H; m), 1.81 (2-H; m), 0.82 (2-Me; d, 7.2); 13C
NMR S 66.9 (1), 46.4 (3), 37.1 (2), 14.4 (2-Me).
To a solution of amino alcohol Y(2.0g, 22mmol) in 39mL of a 10 % solution of triethylamine in MeOH was added di-tert-butyl dicarbonate (5.4g, 25mmo1) and the mixture was stirred at 25 C for 30 min. After removal of solvent, the residue was dissolved in CH2C12 and the solution was washed twice with 1M KHSO4 (pH 2) and once with saturated NaC1 solution, and dried (MgSO4). Removal of solvent in vacuo afforded 4.3g (100% yield) of (R)-3-(tert-butoxycarbonyl)amino-2-methylpropan-l-ol as a viscous oil which was directly used for the next step without further purification (>
95 % pure by NMR analysis); IR P. 3356, 1976, 1686, 1523, 1456 cm'1; 'H NMR 6 4.82 (NH; br s), . 3.54 (1-H; dd, -11.4/4.2), 3.31 (1-H/3-H; m), 3.25 (3-H; dd, -14.1/6.6), 1.77 (2-H; m), 1.44 (CMe3; s), 0.87 (2-Me; d, 6.9).

To a solution of alcohol (R)-3-(tert-butoxycarbonyl)amino-2-methylpropan-l-ol (2.2g, 12mmo1) and sodium periodate (7.5g, 35mmol) in carbon tetrachioride (25mL), acetonitrile (25mL) and water (38mL) was added ruthenium trichloride hydrate (51mg, 0.25mmo1), and the mixture was stirred at 25 C for lh. The mixture was diluted with CH2C12 (100mL) and then filtered through Celite. The filtrate was basified (pH
9) with 2 M K2CO3 solution and the water layer was washed with ether. The aqueous layer was acidified with 1M KHSO4 to pH 2 at 0 C and extracted with CH2C12 (20mL x 3).
The combined extracts ware washed with saturated NaCI solution and dried (MgSO4).
Removal of solvent in vacuo yielded 2.Og (85% yield) of (R)-3-(tert-butoxycarbonyl)amino-2-methylpropanoic acid (Z) as a sticky solid. Pure Z
(1.75g, 74%
yield) crystallized from ether, mp 69.5-70.5 C; [a]D -18.4 (c 2, MeOH); EIMS
m/Z (rel intensity) 147 (60; M+-Me2C=CH2), 130 (12), 74 (29), 57 (100); HREIMS m/z 147.0517 (C5H9NO4, A+1.4mmu); IR v111eJ1 3322-2400, 2797, 1711, 1654, 1413 cm-1; 1H NMR
of major conformer 8 5.00 (NH; br s), 3.32 (3-H; m), 3.24 (3-H'; m), 2.71 (2-H;
m), 1.44 (CMe3; s), 1.20 (2-Me; d); 13C NMR of major/minor (2:1 ratio) conformers S
180.7/179.5 (1), 156.0/157.7 (BOC CO), 79.5/81.0 (CMe3), 42.7/44.0 (3), 39.9/40.2 (2), 28.3/28.3 (CM~e ), 14.6/14.6 (2-Me). Anal. Found: C, 53.04; H, 8.62.
Calcd for C9H17NO4: C, 53.18; H, 8.43.
To a solution of 2.66g of L-leucic acid (20mmo1) and 1.74g of sodium bicarbonate (20mmo1) in 30mL water at 0 C was added 30mL of a CH2C12 solution of 6.44g of tetrabutylammonium chloride (20mmo1) and 1.74mL of allyl bromide (20mmo1).
After vigorously stirring the mixture for 24h, the CH2C12 was evaporated. About 50mL
water was added and the aqueous layer was extracted four times with EtzO. The ether solution was dried over anhydrous sodium sulfate and then evaporated. The residue was passed through a short Si column to give 3.21g of allyl (2S)-2-hydroxy-4-methylpentanoate (AA) (93% yield) as a colorless oil, [a]D -8.4 (c 1.1, CHC13); IR P. 3464, 2957, 1732, 1203, 1140, 1087 cm''; 'H NMR 8 5.92 (allyl 2-H; m), 5.34 (allyl 3-Hz; dd, 17.4/1.1), 5.28 (allyl 3-HE; dd, 10.5/1.1), 4.67 (allyl 1-H2; d, 5.7), 4.23 (2-H; br s), 2.64 (OH; br s), 1.89 (4-H; m), 1.57 (3-H2; m), 0.96 (5-H3; d, 6.5), 0.95 (4-Me; d, 6.7);

175.3 (1), 131.4 (allyl C-2), 118.6 (3), 68.9 (2), 65.7 (allyl C-1), 43.2 (3), 24.1 (4), 23.0 (5), 21.3 (4-Me).

To a solution of 1.74g of Z(8.55mmo1), 1.34g of AA (8.Ommol), and 64mg DMAP in 12mL of dry CH2C12 at 0 C was added dropwise 8mL of a solution of DCC
(2.47g, 12mmol) in CH2C12. The clear solution was stirred at 0 C for 30 min and then at 23 C for 3h. The white precipitate was filtered off, the solvent was evaporated, and the residue was redissolved in Et20. The ether solution was washed successively with cold 0.5N HCI, sodium bicarbonate, and brine. The dried (Na2SO4) ether layer was evaporated and the product was purified by flash column chromatography (silica gel) to give 2.62g (92% yield) of pure allyl (2S,2'R)-2-[3'(tert-butoxycarbonyl)amino-2'-methyl-propanoyl-oxy]-4- methylpentanoate (AB) as a colorless oil, [a]D -51.3 (c 3.41, CHC13);
EIMS m/z (rel intensity) 301 (5.2), 284 (4.0), 258 (1.5), 228 (43.5), 170 (41.8), 130 (74.5), 112 (100); HREIMS m/z 301.1532 (C14H23NO6, A-0.7mmu, M-Me.2C=CH2), 284.1496 (C14H22NO5, A+0.2mmu); IR P. 3395, 2962, 1747, 1715, 1515, 1251, 1175, 1083 cm-'. 'H NMR unit C S 5.17 (NH; br s), 3.42 (3-H; m), 3.22(3-H'; m), 2.78 (2-H, m), 1.43 (CMe3; br s), 1.21 (2-Me; d, 7.1); unit D 8 5.90 (allyl 2-H; m), 5.33 (allyl 3-Hz; d, 16.3), 5.27 (allyl 3-HE; d, 10.3), 5.09 (2-H; dd, 9.7/3.7), 4.63 (allyl 1-H2; m), 1.80 (3-H2; m), 1.64 (4-H; m), 0.96 (5-H3; d, 6.5), 0.94 (4-Me; d, 7.3). 13C
NMR unit C 8 174.7 (1), 156.0 (BOC CO), 79.2 (CMe3), 43.1 (3), 40.3 (2), 28.3 (CMe ), 14.5 (2-Me); unit D 8 170.4 (1), 131.4 (allyl C-2), 119.0 (allyl C-3), 70.9 (2), 65.9 (allyl C-1), 39.6 (3), 24.7 (4), 23.0 (5), 21.5 (4-Me).
To lOmL of a solution of 282mg (0.8mmol) of AB and 91mg (0.08mmo1) of tetrakis(triphenylphosphine)palladium in dry THF was slowly added 688 L
(8mmol) of dry morpholine. After stirring for 40 min, the solvent was evaporated and 100mL of CHZC12 was added. The solution was washed successively with 2N HCI and water.
The organic layer was filtered and the filtrate was extracted twice with saturated sodium bicarbonate. After back-washing with CHZCl2, the aqueous layer was first acidified to pH
3 with cold KHSO4 at 0 C and then extracted three times with ether. The dried ether extract was evaporated to give 250mg of (2S,2'R)-2-[3'(tert-butoxycarbonyl)amino-2'-methylpropanoyl-oxy]-4-methylpentanoic acid (AC) (100% yield) as a wax-like solid, [a]
D-47.9 (c 4.7, CHC13); EIMS m/z (rel intensity) 261 (12), 244 (18), 217 (28), 198 (17), 188 (100), 160 (61); HREIMS m/z 261.1221 (C11H19NO6, A-0.8mmu, M-Me2C=CH2), 244.1221 (C11H18NO5, 0-3.6mmu); IR v,,. 3376, 2960, 1738, 1518, 1174, 786 cm-'. 1H
NMR (CDC13+D20) unit C 6 3.49 (H-3; dd, -13.8/3.5), 3.12(3-H; dd, -13.8/8.7), 2.68 (2-H; m), 1.43 (CMe,; br s), 1.21 (2-Me; d, 7.1); unit D b 5.12 (2-H; dd, 9.6/3.5), 1.90-1.68 (3H/4-H; m), 0.97 (5-H; d, 6.1), 0.94 (4-Me; d, 6.0). 13C NMR unit C 6 174.6 or 174.8 (1), 156.1 (BOC CO), 79.5 (CMe3), 43.0 (3), 40.4 (2), 28.3 (CMez), 14.5 (2-Me);
unit D 6 174.6 or 174.8 (1), 70.5 (2), 39.5 (3), 24.7 (4), 23.0 (5), 21.4 (4-Me).
Compound AD
Alcohol W (34.8mg, 0.056mmo1), compound AC (26.8mg, 0.085mmo1) and DMAP (1.74mg) were dissolved in 2831LL of dichloromethane. To this mixture was added 666 L of dichloromethane solution of DCC (17.5mg, 0.085mmol). The reaction mixture was stirred overnight. The solvent was evaporated with a stream of nitrogen, ether was added, and the white precipitate was filtered off. The filtrate was washed successively with 0.5N HC1, sat. sodium bicarbonate, and brine. Concentration and chromatography (silica column, 25 % EtOAc/hexane) gave 46mg of compound AD (90 %);
[a]D -11.8' (CHC13, c 2.0); IR v,,.3369, 2961, 1737, 1511, 1252, 1174, 1066, 813, 756 cm''. 'H NMR (500MHz) 8 unit A 7.25 (10-H/14-H, dt, 8.5, 2.1), 6.84 (11-H/13-H, dt, 8.5, 2.1), 6.76 (3-H, ddd, 15.5, 6.5, 6.4), 6.34 (8-H, d, 15.6), 5.88 (2-H, bd, 15.5), 5.86 (7-H, dd 15.6, 8.7), 5.04 (5, m), 3.80 (OMe, s), 2.56 (6-H, m), 2.52 (4-H, m), 1.10 (6-Me, d, 6.8); unit B 7.19 (5-H, d, 2.1), 7.05 (9-H, dd, 8.5, 2.1), 6.83 (8-H, d, 8.5), 6.55 (NH, bd, 7.3), 5.04 (2-H, m), 4.78/4.70 (CH2CC13, ABq, -11.8), 3.85 (OCH3, S), 3.19 (3-H, dd, 6.3, -13.8), 3.08 (3-H', dd, 6.8, -13.8); unit C 5.14 (NH, bt, 6.3), 3.32 (3-H, m), 3.20 (3-H', m), 2.73 (2-H, m), 1.42 (CMe3, s), 1.18 (2-Me, d, 7.0); unit D 4.93 (2-H, dd, 10.0, 3.7), 1.67 (3-H/4-H, m), 1.55 (3-H', m), 0.86 (5-H, d, 6.5), 0.83 (4-Me-H, d, 6.5). 13C NMR 6 unit A 165.4 (1), 159.1 (12), 139.3 (3), 131.1 (9), 129.7_(7), 128.5 (8), 127.3 (10, 14), 125.4 (2), 114.0 (11, 13), 76.5 (5), 55.3 (OMe), 41.1 (6), 33.4 (4), 16.7 (6-Me); unit B 170.5 (1), 154.1 (7), 131.2 (5), 128.9 (4), 128.5 (9), 122.4 (6), 112.1 (8), 94.3 (CC13), 74.6 (CH2CCl3), 56.1 (OMe), 53.2 (2), 36.6 (3);
unit C 175.2 (1), 156.0 (BOCCO), 79.3 (CMe3), 43.1 (3), 40.4 (2), 28.3 (CMe=), 14.4 (2-Me); unit D 170.1 (1), 71.4 (2), 39.5 (3), 24.7 (4), 22.9 (5), 21.4 (4-Me).
Cryptophycin 81 Compound AD (46mg, 0.05mmo1) was mixed with activated Zn dust (178mg, 30 excess) in 1.3mL HOAc. The mixture was subjected to ultrasonication for 45 minutes, and then stirred for another 90 minutes. About 30mL of dichloromethane was added.
The solid was filtered off and the filtrate was evaporated under vacuum. The residue was dissolved in 1.1mL of TFA and the solution wasstirred for one hour. TFA was evaporated under vacuum and water was added. Lyophilization gave the free amino acid.
The amino acid was dissolved in 4.6mL DMF. To this solution was added 26 L
DIEA
and FDPP (30mg, 0.075mmol, in 2.2mL DMF), respectively. After stirring for 6 hours, the solvent was evaporated and EtOAc was added. The solution was washed with 0.5N
HCI, and brine respectively. Evaporation of solvent followed by chromatographic purification (Si, ether) produced 20.5mg of Cryptophycin 81 (61 %); [a]D +34.9 (CHC13, c 0.45); IR v,,= 3409, 3270, 2958, 1746, 1725, 1672, 1511, 1251, 1175, 1066, 1025, 972, 816 cm'. 'H NMR (500 MHz) S unit A 7.26 (10-H/14-H; dt, 8.6, 2.5), 6.84(11-H/13-H, dt, 8.6, 2.5), 6.68 (3-H, ddd, 15.4, 9.9, 5.6), 6.35 (8-H, d, 15.9), 5.86 (7-H, dd, 15.9, 8.8), 5.77(2-H, dd, 15.4, 0.9), 4.99 (5-H, ddd, 11.2, 6.0, 1.7), 3.80 (OCH3, S), 2.53 (4-H/6-H, m), 2.37 (4-H', ddd, 11.2, 9.9,-14.6), 1.12 (6-Me, d, 6.9);
unit B
7.22 (5-H, d, 2.2), 7.08 (9-H, dd, 8.4, 2.4), 6.84 (8-H, d, 8.4), 5.64 (NH, d, 8.6), 4.81 (2-H, m), 3.86 (OMe, S), 3.13 (3-H; dd, 5.6, -14.5), 3.05 (3-H', dd, 7.1, -14.5); unit C, 6.93 (NH, bdd, 5.8, 5.6), 3.50 (3-H, ddd, 5.2, 3.9, -13.5), 3.28 (3-H', ddd, 6.9, 6.7, -13.5), 2.71 (2-H, m), 1.22 (2-Me; d, 7.3); unit D 4.84 (2-H, dd, 10.1, 3.4), 1.67 (3-H/
4-H; m), 1.38 (3-H', m), 0.78 (5-H, d, 6.5), 0.75 (4-Me-H, d, 6.5). 13C NMR
(125 MHz) 8 unit A 165.4 (1), 159.2 (12), 141.4 (3), 131.1 (9), 129.6 (7), 128.4 (8), 127.3 (10, 14), 125.2 (2), 114.1 (11, 13), 77.5 (5), 55.3 (OMe), 42.2 (6), 36.4 (4), 17.4 (6-Me); unit B 171.0 (1), 154.0 (7), 131.2 (5), 129.9 (4), 128.4 (9), 122.5 (6), 112.1 (8), 56.2 (OMe), 53.5 (2), 35.1 (3); unit C 175.6 (1), 41.2 (3), 38.3 (2), 14.0 (2-Me); unit D
170.9 (1), 71.6 (2), 39.5 (3), 24.5 (4), 22.7 (5), 21.3 (4-Me).

Exar_nple 15 Synthesis of Cryptophvcin 82 Compound AE
Compound AE is the tert-butyldiphenylsilyl ether (TBDMS) derivative of E.
Compound AF
The hydrolysis of AE (150mg) to AF (125mg) was carried out in 87% yield using the procedure described above for the hydrolysis of T to U. 1H NMR 5 7.69/7.64 (SiPh2, 2'-H, 6'-H/2"-H, 6"-H, d, 6.3), 7.41 (SiPh2, 4'-H/4"-H, m), 7.39 (SiPh2, 3'-H, 4-Me-H/
3"-H, 5"-H, m), 6.86 (3-H; dt; 15.5, 7.5), 5.62 (2-H, d, 15.5), 5.30 (7-H/8-H, m), 3.72 (5-H, m), 2.28 (4-H, m), 2.20 (6-H, m), 1.52 (9-H, bd, 7.7), 1.08 (CMe3, s), 1.00 (6-CH3, d, 6.7).
Compound AG

The preparation of AG (96mg) from AF (76mg, 0.18mmo1) was carried out in 5 70%
yield using the procedure described above for the preparation of V from U. 1H

NMR 8 unit A 7.67 (SiPh2, 2'-H, 6'-H/2"-H, 6"-H; m), 7.46--7.31 (SiPh2, 3'-H, 4'-H, 4-Me-H/3"-H, 4"-H, 5"-H, m), 6.62 (3-H, dt, 15.4, 7.6), 5.51/5.48 (2-H, d, 15.4), 5.33 (7/8, m), 3.70 (5-H, m), 2.22 (4-H/6-H, m), 1.61 (9, bd, 7.4), 1.06 (CMe3, S), 0.98 (6-Me, d, 6.8); unit B 7.16 (5-H, d, 1.7), 7.00 (9-H, dd, 8.5, 1.7), 6.83/6.82 (8-H, d, 8.5), 5.68/5.66 (NH, d, 7.3), 5.03 (2-H, m), 4.78/4.73 (CH2CC13, ABq, -11.9), 3.87 (OMe, S), 3.16 (3-H; m), 3.09 (3-H', m). 13C NMR 8 unit A 165.1 (1), 143.0/142.9 (3), 136.0 (SiPh2, 2', 6'/2 ', 6"), 134.3/133.7 (SiPh2, i'/1"), 132.5 (7), 129.6 (8), 129.6 (SiPh2, 4'/4"), 127.5 (SiPh2, 3', 4-Me/3", 5"), 125.7/125.4 (2), 76.3 (5), 41.6 (6), 36.9/36.8 (4), 27.0 (CMe=), 19.5 (CMe3), 18.1 (9), 16.4/16.3 (6-Me); unit B
170.0 (1), 154.2 (7), 131.0 (5), 128.4 (4/9), 122.5 (6), 112.1 (8), 94.2 (CC13), 74.7 (CHZCCI3), 56.1 (OMe), 52.9 (2) 36.4 (3).
Compound AH

The preparation of AH (53.4mg) from AG (84mg, 0.llmmol) was carried out in 92% yield using the procedure described above for the preparation of W from V.

NMR 8 unit A 6.83 (3-H, dt, 15.4, 7.5), 5.80 (2-H, d, 15.4), 5.51 (8-H, m), 5.33 (7-H, dd, 15.2, 8.3), 3.50 (5-H, m), 2.42 (4-H, m), 2.30 (4-H', m), 2.28 (6, m), 1.68 (9, d, 7.3), 0.99 (6-Me, d, 6.7); unit B 7.20 (5-H, d, 1.8), 7.03 (9-H, dd, 8.4, 1.8), 6.80 (8-H, d, 8.4), 6.10 (NH, bd, 7.0), 5.07 (2-H, m), 4.80/4.70 (CH2CC13, ABq, -11.5), 3.88 (OCH3, S), 3.20 (3-H, dd, 5.5, -14.3), 3.10 (3-H', dd, 7.2, -14.3). 13C NMR S
unit A
165.4 (1), 142.8 (3), 132.2 (7), 127.7 (8), 125.7 (2), 73.6 (5), 42.8 (6), 36.9 (4), 18.1 (9), 16.8 (6-Me); unit B 170.2 (1), 154.2 (7), 131.0 (5), 128.5 (4/9), 122.5 (6), 112.2 (8), 94.2 (CC13), 74.7 (CH2CC13), 56.1 (OMe), 53.1 (2), 36.5 (3).
Cr,yptophycin 82 Compound Al (36.8mg, 86% yield) was prepared from 27.4mg (0.052mmo1) of compound AH using the procedure described above for AD.
Compound Al (68mg, 0.083mmo1) was cyclized to Cryptophycin 82 (28.5mg) using the procedure described above for the cyclization of AD to Cryptophycin 81; [a]D

+19.9 (CHC13, c 2.0). 'H NMR (500 MHz) S unit A 6.65 (3-H, ddd, 15.4, 9.5, 5.6), 5.76 (2-H, d, 15.4), 5.48 (8-H, dq, 15.3, 6.5), 5.27 (7-H, ddd, 15.3, 8.4, 1.5), 4.89 (5-H, ddd, 10.7, 5.6, 1.5), 2.38 (4-H, m), 2.33 (4-H'/6-H, m), 1.66 (9-H, dd, 6.4, 1.5), 1.00 (6-Me, d, 6.9); unit B 7.22 (5-H, d, 2.0), 7.08 (9-H, dd, 8.4, 2.0), 6.83 (8-H, d, 8.4), 5.74 (NH, m), 4.81 (2-H, ddd, 8.5, 7.3, 5.6), 3.87 (OMe, S), 3.13 (3-H;
dd, 5.6, -14.4), 3.04 (3-H', dd, 7.3, -14.4); unit C, 6.93 (NH, bdd, 6.7, 4.7), 3.52 (3-H, ddd, 4.7, 3.9, -13.5), 3.27 (3-H', ddd, 6.8, 6.7, -13.5), 2.72 (2-H, dqd, 7.1, 6.8, 3.9), 1.20 (2-Me; d, 7.1); unit D 4.88 (2-H, dd, 9.6, 3.4), 1.75 (3-H/ 4-H; m), 1.47 (3-H', m), 0.94 (5-H, d, 6.2), 0.91 (4-Me-H, d, 6.4). 13C NMR (125 MHz) b unit A 165.5 (1), 141.7 (3), 131.4 (7), 127.1 (8), 125.2 (2), 77.7 (5), 41.5 (6), 36.2 (4), 17.9 (9), 17.2 (6-Me); unit B 171.0 (1), 153.9 (7), 131.1 (5), 129.9 (4), 128.4 (9), 122.4 (6), 112.2 (8), 56.1 (OMe), 53.5 (2), 35.1 (3); unit C 175.5 (1), 41.3 (3), 38.2 (2), 14.0 (2-Me); unit D
170.9 (1), 71.5 (2), 39.6 (3), 24.6 (4), 23.0 (5), 21.5 (4-Me).

Example 16 Synthesis of CrYptophycin 90 and Crvptophvcin 91 General Procedure for Epoxidation of S rene-tvne Cryptophycins To a solution of the cryptophycin (about lOmg/mL) in dichloromethane was added three equivalents of m-chloroperbenzoic acid in dichloromethane (about lOmg/mL). The solution was stirred at room temperature until all starting material was consumed. The solution was passed through a short silica column using CH2C12 and then 1:4 CH2C12: EtZ0 to give a mixture of the two epoxides. The epoxides were separated by HPLC (C-18, 7:3 MeCN:H20).
Using this procedure, Cryptophycin 82 (5mg) was converted to 2.2mg of Cryptophycin 90 and 1.2mg of Cryptophycin 91.
Spectral Data for Cryptoph cin 90 'H NMR (500 MHz) S unit A 6.67 (3-H, ddd, 15.4, 9.5, 5.8), 5.79 (2-H, d, 15.4), 5.09 (5-H, ddd, 10.4, 4.5, 2.6), 2.84 (8-H, qd, 5.2, 2.2), 2.57 (7-H, dd, 7.6, 2.2), 2.46 (4-H, m), 1.82 (6-H, m), 1.32 (9-H, d, 5.2), 1.03 (6-Me, d, 6.9);
unit B 7.22 (5-H, d, 2.2), 7.08 (9-H, dd, 8.4, 2.2), 6.84 (8-H, d, 8.4), 5.74 (NH, d, 8.6), 4.81 (2-H, ddd, 8.3, 7.4, 5.7), 3.87 (OMe, S), 3.14 (3-H; dd, 5.4, -14.5), 3.03 (3-H', dd, 7.3, -14.5); unit C, 6.95 (NH, bdd, 6.7, 4.8), 3.52(3-H, ddd, 4.8, 3.7, -13.4), 3.29 (3-H', ddd, 6.7, 6.5, -13.4), 2.74 (2-H, dqd; 7.3, 6.5, 3.7), 1.24 (2-Me; d, 7.3);
unit D 4.90 (2-H, dd, 9.9, 3.7), 1.75 (3-H, m), 1.60 (4-H; m), 1.49 (3-H', ddd, 8.7, 3.7, -13.7), 0.95 (5-H, d, 6.5), 0.91 (4-Me-H, d, 6.7). 13C NMR (125 MHz) 6 unit A 165.4 (1), 141.1 (3), 125.3 (2), 76.3 (5), 60.0 (7), 56.8 (8), 40.2 (6), 36.5 (4), 17.5 (9), 13.3 (6-Me); unit B 171.0 (1), 154.0 (7), 131.1 (5), 129.9 (4), 128.4 (9), 122.5 (6), 112.3 (8), 56.1 (OMe), 53.6 (2), 35.1 (3); unit C 175.5 (1), 41.2 (3), 38.3 (2), 14.1 (2-Me); unit D
170.7 (1), 71.4 (2), 39.6 (3), 24.7 (4), 23.0 (5), 21.5 (4-Me) Spectral Data for Cryptophycin 91 'H NMR 6 unit A 6.68 (3-H, ddd, 15.4, 9.8, 5.4), 5.78 (2-H, d, 15.4), 5.09 (5-H, ddd, 11.1, 3.7, 2.0), 2.75 (8-H, m), 2.62 (7-H, dd, 9.5, 2.1), 2.48 (4-H, m), 1.78 (6-H, m), 1.32 (9-H, d, 5.2), 0.99 (6-Me, d, 7.1); unit B 7.23 (5-H, d, 1.9), 7.09 (9-H, dd, 8.4, 1.9), 6.85 (8-H, d, 8.4), 5.68 (NH, d, 8.4), 4.82 (2-H, m), 3.88 (OMe, S), 3.15 (3-H; dd, 5.4, -14.4), 3.04 (3-H', dd, 7.2, -14.4); unit C, 6.95 (NH, bdd, 6.0, 4.6), 3.53(3-H, ddd, 5.0, 4.0, -13.4), 3.29 (3-H', ddd, 6.9, 6.7, -13.4), 2.75 (2-H, m), 1.23 (2-Me;
d, 6.7); unit D 4.92 (2-H, dd, 9.8, 3.4), 1.77 (3-H, m), 1.58 (4-H; m), 0.96 (5-H, d, 7.3), 0.92 (4-Me-H, d, 6.7).

Example 17 Synthesis of Cryptophycin 97 To a solution of the cyclic depsipeptide, Cryptophycin 53 (9mg, 0.013mmo1), dissolved in dimethyl sulfoxide (lmL), was added sodium azide (40mg) and concentrated sulfuric acid (4 L). The mixture was then allowed to stir at 75-85 C for 2 days. After this time the reaction mixture was cooled to room temperature, diluted with Et20 (15mL) and the organic layer washed with brine (2 x 20mL) and H20 (20mL). The ethereal extract was then dried (MgSO4) and the solvent removed in vacuo to return an amorphous, colorless solid, that was predominantly the azido-alcohol, Cryptophycin 86. Purification was achieved by reverse phase chromatography (ODS, 101L, 250 x 10mm, 25% H20/MeCN, 3mL min71) to return the pure azido-alcohol, Cryptophycin 86, as an amorphous colorless solid (7.4mg, 77%).
Spectral Data for Crvytophycin 86 [a]D -22.0 (c=3.0, CHC13); MS (EI) m/z 482/484 (highest observed ion, M+-229, 8/3), 625/627 (14/5), 259 (7), 195/197 (100/34), 184 (14), 155/157 (82/70), 91 (23), 77 (22); HRMS, obsd mlz 482.1791, C23H31N2O735C1 (A 2.9mmu); MS (FAB) m/z (magic bullet matrix) 712/714 (M++H, 79/36), 686/688 (31/12), 232 (74), 184 (100). 'H
NMR

(CDC13) 8 unit A: 7.37-7.42 (10/11/12/13/14-H, bm, W1,z =20), 6.77 (3-H, ddd, 15.2, 10.6, 4.4), 5.77 (2-H, dd, 15.2, 1.3), 5.45 (5-H, ddd, 11.0, 4.2, 2.0), 4.55 (8-H, d, 5.7), 3.75 (7-H, dd, 7.3, 5.7), 2.55 (4-Hb, dddd, 14.5, 4.4, 2.0, 1.3), 2.43 (4-Ha, ddd, 14.5, 11.0, 10.6), 2.34 (7-OH, s), 1.80 (6-H, ddq, 7.3, 4.2, 7.0), 0.99 (6-Me, d, 7.0), unit B: 7.20 (5-H, d, 2.2), 7.06 (9-H, dd, 8.4, 2.2), 6.84 (8-H, d, 8.4), 5.76 (NH, d, 7.7), 4.74 (2-H, ddd, 7.7, 7.5, 5.5), 3.87 (OCH3, s), 3.10 (3-Hb, dd, 14.5, 5.5), 3.06 (3-Ha, dd, 14.5, 7.5), unit C. 7.22 (NH, dd, 8.4, 3.7), 3.40 (3-Hb, dd, 13.6, 8.4), 3.14 (3-Ha, dd, 13.6, 3.7), 1.23 (2-CH3, s), 1.16 (2-CH3', s), unit D: 4.85 (2-H, dd, 9.5, 5.1), 1.71 (3-Hb, ddd, 13.6, 9.5, 5.9), 1.59 (4-H, bm, Wln = 25), 1.50 (3-H$, ddd, 13.6, 7.9, 5.1), 0.89 (4-CH3, d, 6.6), 0.85 (5-H3, d, 6.6); 13C NMR (CDC13) S unit A:
165.3 (1), 143.0 (3), 135.1 (9), 129.1 (12), 128.9 (11/13), 128.6 (10/14), 124.3 (2), 75.1 (7), 74.6 (5), 67.8 (8), 39.3 (6), 34.7 (4), 11.9 (6-Me), unit B: 170.5 (1), 154.1 (7), 130.9 (5), 129.7 (4), 128.3 (9), 122.5 (6), 112.4 (8), 56.1 (7-OMe), 54.3 (2), 35.3 (3), unit C:
177.8 (1), 46.5 (3), 42.8 (2), 22.9 (2-Me), 22.7 (2-Me'), unit D: 170.2 (1), 71.4 (2), 39.4 (3), 24.7 (4), 22.6 (4-Me), 21.8 (5).
Cryptophycin 97 To a solution of the cyclic depsipeptide, Cryptophycin 86 (5.5mg, 0.008mmo1), dissolved in Et20/CH2C12 (3:1, 0.5mL), was added an ethereal solution (0.5mL) of triphenylphosphine (3mg, 0.011mmo1). The mixture was then allowed to stir at room temperature for three days. After this time the solvent was removed in vacuo and the residue dissolved in CH2C12 and subject to HPLC purification (CN column, 10 , 250 x 10mm, 80% EtOAc/CH2C12, 3mL min'') to return pure Cryptophycin 97 as an amorphous colorless solid (4.2mg, 82 %).
UV (MeOH) Xn. (e) 202 (24400), 218 (9400), 284 (2200) nm; MS (EI) m/z 667/669 (M+, 11/3), 639/641 (41/16), 442 (21), 226 (17), 195 (43), 196 (32), 197 (100), 198 (71), 199 (11), 182/184 (25/16), 155/157 (63/22), 146 (30), 91 (40), 77 (29);
HRMS, obsd m/z 667.3061, C36H46N30735C1 (0-3.6mmu). 'H NMR (CDC13) 6 unit A 7.35 (11-H/13-H; m, W1n=15 Hz), 7.28 (12-H, m), 7.16 (10-H/14-H; m, W1,2-:=15 Hz), 6.74 (3-H; ddd, 15.2/9.0/6.1), 5.69 (2-H; d, 15.2), 5.21 (5-H; ddd, 9.2/4.3/4.2), 2.79 (8-H, bs), 2.51 (4-H2, m), 2.11 (7-H, bd, 6.5), 1.48 (6-H, m), 1.13 (6-Me, d, 6.9); unit B: 7.18 (5-H, d, 2.1), 7.04 (9-H, dd, 8.4, 2.1), 6.83 (8-H, d, 8.4), 5.53 (NH, m), 4.73 (2-H, ddd, 7.6, 5.6, 5.4), 3.87 (OMe, s), 3.09 (3-Hb, dd, 14.7, 5.4), 3.04 (3-Ha, dd, 14.7, 7.6), unit C.- 7.20 (NH, m), 3.40 (3-Hb, dd, 13.5, 8.6), 3.11 (3-Ha, dd, 13.5, 3.3), 1.22 (2-Me, s), 1.15 (2-Me', s), unit D: 4.84 (2-H, dd, 10.1, 3.7), 1.71 (3-Hb, m), 1.67 (4-H, bm, W1,2 = 25), 1.35 (3-H~, m), 0.86 (4-Me, d, 6.7), 0.84 (5-H3, d, 6.5); 13C
NMR
(CDC13) 6 unit A: 165.0 (1), 142.1 (3), 139.2 (9), 128.8 (11/13), 127.5 (12), 125.4 (10/14), 124.6 (2), 76.6 (5), 42.5 (6), 42.1 (7), 40.5 (8), 36.6 (4), 14.8 (6-Me), unit B:
170.2 (1), 154.1 (7), 130.9 (5), 129.5 (4), 128.2 (9), 122.6 (6), 112.4 (8), 56.1 (7-OMe), 54.3 (2), 35.3 (3), unit C: 177.9 (1), 46.5 (3), 42.7 (2), 22.8 (2-Me/2-Me'), unit D: 170.5 (1), 71.3 (2), 39.5 (3), 24.6 (4), 22.7 (4-Me), 21.3 (5).

Example 18 Synthesis of Cryptophycins 110-112 and 124 Cryptophycin 108 To a mixture of Cryptophycin 90 and Cryptophycin 91 (27mg, 0.045mmo1) in 0.8mL of tetrahydrofuran was added 400 L of an aqueous solution of periodic acid (32mg, 0.14mmo1). The clear solution was stirred at room temperature for 5 hours.
Water was added and the aqueous layer was extracted twice with ethyl acetate.
The organic layer was washed with water, dried and concentrated. The residue was purified by reversed-phase chromatography on an ODS column (1:1 MeCN:H20) to give a 90%
yield of Cryptophycin 108. 'H NMR (500 MHz) 6 unit A 9.64 (7-H, d, 1.9), 6.67 (3-H, ddd, 15.3, 10.0, 5.4), 5.81 (2-H, dd, 15.3, 0.9), 5.32 (5-H, ddd, 11.0, 6.6, 2.1), 2.65 (6-H, qdd, 7.2, 6.9, 1.9), 2.53 (4-H, m), 2.44 (4-H', m), 1.17 (6-Me, d, 7.2);
unit B
7.21 (5-H, d, 2.2), 7.08 (9-H, dd, 8.4, 2.2), 6.84 (8-H, d, 8.4), 5.91 (NH, d, 8.4), 4.80 (2-H, m), 3.86 (OMe, S), 3.16 (3-H; dd, 5.4, -14.6), 3.00 (3-H', dd, 7.8, -14.6); unit C
7.05 (NH, bdd, 6.9, 5.0), 3.47(3-H, ddd, 4.6, 4.2, -13.5), 3.32 (3-H', ddd, 6.9, 6.6, -13.5), 2.73 (2-H, m), 1.23 (2-Me; d, 7.2); unit D 4.83 (2-H, dd, 9.5, 3.6), 1.75 (3-H, m), 1.70 (4-H; m), 1.40 (3-H', ddd, 9.5, 3.9, -14.0), 0.93 (5-H, d, 6.6), 0.88 (4-Me-H, d, 6.6). 13C NMR (125 MHz) 6 unit A 200.6 (7), 165.4 (1), 140.5 (3), 125.6 (2), 73.7 (5), 50.1 (6), 36.1 (4), 10.8 (6-Me); unit B 171.1 (1), 154.0 (7), 131.0 (5), 129.9 (4), 128.3 (9), 122.4 (6), 112.3 (8), 56.1 (OMe), 53.8 (2), 35.0 (3); unit C 175.6 (1), 41.0 (3), 38.1 (2), 14.1 (2-Me); unit D 170.4 (1), 71.3 (2), 39.3 (3), 24.6 (4), 22.9 (5), 21.4 (4-Me).
Cryptophycin 108 was also produced by selective ozonolysis of Cryptophycin 82 using the procedure described above for the ozonolysis of E to F.

General Procedure for the Wittig Reaction Butyl lithium (0.4mL, 2.5 M in hexane, lmmol) was added to a 10mL solution of the aryltriphenylphosphonium chloride (immol) in THF at -78 C. The reaction mixture was stirred for 15 minutes at -78 C and then was placed in a ice-bath for one hour.
Three equivalents of the above mixture was added to the THF solution of Cryptophycin 108 (about 30mg/mL) at -78 C. The solution was stirred for 20 minutes before the cold bath was removed. When the temperature had risen to 25 C, the reaction was quenched with saturated ammonium chloride aqueous solution. The mixture was extracted twice with ethyl acetate. The organic extract was washed with water, dried and concentrated.
The residue was purified on an ODS flash column (65:35 MeCN:H20) to give a mixture of E and Z isomers (contains 8% to 13 % Z isomers depending on the nature of the aryl group; analysis determined by NMR). The desired E isomer crystallized from ether.
Cryptophvcin 110 The Wittig reaction led to 5. lmg of the p-fluorophenyl analog (contained about 8% Z isomer) from 7.3mg of Cryptophycin 108 (about lmg unreacted aldehyde was recovered). After crystallization from ether, 4.0mg of pure Cryptophycin 110 was obtained; [a]D +42.4 (MeOH, c 2.1). 'H NMR 6 unit A 7.29 (10-H/14-H; dd, 8.6, 5.6), 6.99(11-H/13-H, dt, 8.6, 8.5), 6.68 (3-H, ddd, 15.3, 9.7, 5.6), 6.38 (8-H, d, 15.8), 5.83 (7-H, dd, 15.8, 8.8), 5.78 (2-H, d, 15.3), 5.00 (5-H, ddd, 10.8, 7.3, 1.3), 2.53 (4-H/6-H, m), 2.36 (4-H', m), 1.13 (6-CH3, d, 6.8); unit B 7.21 (5-H, d, 1.8), 7.07 (9-H, dd, 8.4, 1.8), 6.84 (8-H, d, 8.4), 5.68 (NH, d, 8.5), 4.82 (2-H, m), 3.87 (OMe, s), 3.14 (3-H; dd, 5.6, -14.4), 3.04 (3-H', dd, 7.2, -14.4); unit C, 6.95 (NH, bdd, 6.8, 5.9), 3.50 (3-H, td, 4.4, -13.5), 3.28 (3-H', ddd, 6.8, 6.7, -13.5), 2.72 (2-H, m), 1.23 (2-Me; d, 7.2); unit D 4.82 (2-H, m), 1.65 (3-H/4-H; m), 1.35 (3-H', ddd, 4.5, 3.8, -10.9), 0.78 (5-H, d, 6.4), 0.74 (4-Me-H, d, 6.4). 13C NMR (75 MHz) S unit A
165.4 (1), 162.3 (12, d, 'Jc-F 245.8 Hz), 141.4 (3), 132.9 (9), 130.6 (7), 129.9 (8), 127.6 (10/14, d, 3Jc-F 8.0 Hz), 125.2 (2), 115.5 (11/13, d, 2Jc-F 21.5 Hz), 77.4 (5), 42.2 (6), 36.4 (4), 17.3 (6-Me); unit B 170.9 (1), 153.9 (7), 131.0 (5), 129.9 (4), 128.4 (9), 122.4 (6), 112.2 (8), 56.1 (OMe), 53.6 (2), 35.1 (3); unit C 175.6 (1), 41.1 (3), 38.3 (2), 14.0 (2-Me); unit D 170.9 (1), 71.5 (2), 39.5 (3), 24.5 (4), 22.7 (5), 21.2 (4-Me).
, Cryptophycin 111 The Wittig reaction led to 43mg of the p-tolyl analog (contained about 9% Z
isomer) from 55mg of Cryptophycin 108. After crystallization from ether, 34mg of pure Cryptophycin 111 was obtained; [a]D +44.3' (CHC13, c 0.6); EIMS m/z (relative intensity %) 652 (2.9, M+), 497 (3.6), 412 (23.8), 242 (20), 145 (46), 105 (75); high-resolution EIMS 652.29094 (calcd for C36H45C1N207, A-0.6mmu, M+). 'H NMR 6 unit A
7.21 (10-H/14-H; d, 8.0), 7.11 (11-H/13-H, d, 8.0), 6.68 (3-H, ddd, 15.2, 9.6, 5.4), 6.37 (8-H, d, 15.8), 5.95 (7-H, dd, 15.8, 8.6), 5.76 (2-H, d, 15.2), 4.99 (5-H, dd, 10.4, 6.2), 2.51 (4-H/6-H, m), 2.38 (4-H', m), 2.32 (12-Me, s), 1.13 (6-CH3, d, 6.8); unit B
7.22 (5-H, d, 2.1), 7.08 (9-H, dd, 8.4, 2.1), 6.83 (8-H, d, 8.4), 5.80 (NH, d, 8.4), 4.81 (2-H, m), 3.86 (OMe, s), 3.14 (3-H; dd, 5.6, -14.4), 3.03 (3-H', dd, 7.3, -14.4); unit C
6.98 (NH, bdd, 6.0, 5.7), 3.49 (3-H, td, 4.6, -13.4), 3.29 (3-H', ddd, 6.7, 6.6, -13.4), 2.70 (2-H, m), 1.22 (2-Me; d, 7.2); unit D 4.81 (2-H, m), 1.65 (3-H/4-H; m), 1.37 (3-H', m), 0.78 (5-H, d, 5.8), 0.73 (4-Me-H, d, 6.4). 13C NMR (75 MHz) 8 unit A
165.5 (1), 141.5 (3), 137.4 (12), 133.9 (9), 131.7 (7), 129.3 (10/14), 129.0 (8), 126.0 (11/13), 125.1 (2), 77.4 (5), 42.2 (6), 36.4 (4), 21.1 (12-Me), 17.3 (6-Me); unit B
171.0 (1), 153.9 (7), 131.0 (5), 129.9 (4), 128.4 (9), 122.4 (6), 112.2 (8), 56.1 (OMe), 53.6 (2), 35.1 (3); unit C 175.6 (1), 41.1 (3), 38.3 (2), 14.1 (2-Me); unit D 170.9 (1), 71.6 (2), 39.5 (3), 24.5 (4), 22.7 (5), 21.2 (4-Me).
CrYptophvcin 112 The Wittig reaction led to 35mg of the 2-thienyl analog (contained about 13% Z
isomer) from 51mg of Cryptophycin 108. After crystallization from ether, 25mg of pure Cryptophycin 111 was obtained. 'H NMR (500 MHz) S unit A 7.12 (12-H; d, 4.9), 6.94 (11-H, dd, 4.9, 3.4), 6.90 (10-H, d, 3.4), 6.68 (3-H, ddd, 15.2, 9.5, 5.3), 6.54 (8-H, d, 15.7), 5.83 (7-H, dd, 15.7, 8.7), 5.78 (2-H, d, 15.2), 4.96 (5-H, dd, 9.5, 6.5), 2.51 (4-H/6-H, m), 2.35 (4-H', m), 1.13 (6-CH3, d, 6.8); unit B 7.21 (5-H, d, 1.6), 7.07 (9-H, dd, 8.4, 1.6), 6.84 (8-H, d, 8.4), 5.74 (NH, d, 7.1), 4.82 (2-H, m), 3.87 (OMe, s), 3.13 (3-H; dd, 5.5, -14.4), 3.04 (3-H', dd, 7.1, -14.4); unit C, 6.97 (NH, bt, 5.8), 3.50 (3-H, ddd, 4.4, 4.3, -13.4), 3.28 (3-H', ddd, 6.8, 6.6, -13.4), 2.71 (2-H, m), 1.22 (2-Me; d, 7.2); unit D 4.82 (2-H, m), 1.67 (3-H/4-H; m), 1.39 (3-H', m), 0.80 (5-H, d, 6.4), 0.77 (4-Me-H, d, 6.4).

Cryptophvcin 124 The Wittig reaction led to 131mg of the p-chlorophenyl analog (contained about 10% Z isomer) from 153mg of Cryptophycin 108. After crystallization from ether, 107mg of pure Cryptophycin 124 was obtained; [a]D +29.2 (CHC13, c 0.5); high-resolution EIMS m/z 672.23691 (calcd for C35H42C12N2O7, 0 O.Ommu). 'H NMR (500 MHz) 8 unit A 7.26 (10-H/11-H/13-H/14-H, s), 6.68 (3-H, ddd, 15.2, 9.6, 5.4), 6.36 (8-H, d, 15.8), 5.98 (7-H, dd, 15.8, 8.8), 5.77 (2-H, d, 15.2), 5.00 (5-H, bdd, 9.4, 6.3), 2.54 (4-H/6-H, m), 2.38 (4-H', m), 1.13 (6-CH3, d, 6.8); unit B 7.22 (5-H, d, 1.7), 7.07 (9-H, dd, 8.4, 1.7), 6.84 (8-H, d, 8.4), 5.73 (NH, bd, 7.8), 4.82 (2-H, m), 3.86 (OMe, s), 3.13 (3-H; dd, 5.5, -14.4), 3.04 (3-H', dd, 7.2, -14.4); unit C, 6.97 (NH, bt, 5.8), 3.49 (3-H, td, 4.2, -13.4), 3.29 (3-H', ddd, 6.7, 6.6, -13.4), 2.71 (2-H, m), 1.22 (2-Me;
d, 7.2); unit D 4.81 (2-H, m), 1.65 (3-H/4-H; m), 1.35 (3-H', m), 0.77 (5-H, d, 7.1), 0.75 (4-Me-H, d, 7.1). 13C NMR (75 MHz) 8 unit A 165.4 (1), 141.3 (3), 135.2 (12), 133.2 (9), 130.9 (7), 130.6 (8), 128.7 (11/13), 127.3 (10/14), 125.2 (2), 77.3 (5), 42.2 (6), 36.5 (4), 17.3 (6-Me); unit B 170.9 (1), 153.9 (7), 131.0 (5), 129.8 (4), 128.4 (9), 122.4 (6), 112.2 (8), 56.1 (OMe), 53.6 (2), 35.1 (3); unit C 175.6 (1), 41.1 (3), 38.3 (2), 14.1 (2-Me); unit D 170.8 (1), 71.5 (2), 39.6 (3), 24.5 (4), 22.8 (5), 21.3 (4-Me).
Example 19 Synthesis of Cryptophycins 115-120. 125 and 126 Cryptophycins 115 and 116 Using the general procedure described above for epoxidation of styrene-type cryptophycins, Cryptophycin 110 (3.5mg) was converted to 2.0mg of Cryptophycin and lmg of Cryptophycin 116.
Spectral Data for Cryptophycin 115 [a]D +29.1 (MeOH, c 0.8); EIMS m/z (relative intensity %) 672 (1.9, M+), 412 (5.8), 245 (17), 195 (52), 155 (31), 141 (23), 135 (15), 109 (100); high-resolution EIMS
668.2853 (calcd for C35H42C1FN208, A+3.4mmu, M+). 'H NMR (500 MHz) 6 unit A
7.22 (10-H/14-H; ddt, 8.7, 5.2, 2.0), 7.01 (11-H/13-H, ddt, 8.7, 8.5, 2.0), 6.68 (3-H, ddd, 15.2, 9.7, 5.2), 5.74 (2-H, dd, 15.2, 0.8), 5.15 (5-H, ddd, 11.2, 5.0, 1.8), 3.67 (8-H, d, 2.0), 2.88 (7-H, dd, 7.4, 2.0), 2.54 (4-H, dtd, 5.2, 1.8, -14.4), 2.44 (4-H', ddd, 11.2, 9.7, -14.4), 1.79 (6-H, m), 1.13 (6-CH3, d, 6.8); unit B 7.21 (5-H, d, 2.0), 7.06 (9-H, dd, 8.3, 2.0), 6.83 (8-H, d, 8.3), 5.63 (NH, d, 8.4), 4.80 (2-H, ddd, 8.4, 7.2, 5.4), 3.87 (OMe, s), 3.14 (3-H; dd, 5.4, -14.4), 3.03 (3-H', dd, 7.2, -14.4);
unit C 6.94 (NH, bdd, 6.7, 5.0), 3.48 (3-H, ddd, 5.0, 3.7, -13.4), 3.30 (3-H', ddd, 6.8, 6.7, -13.4), 2.72 (2-H, m), 1.22 (2-Me; d, 7.4); unit D 4.83 (2-H, dd, 9.9, 3.6), 1.70 (3-H/4-H; m), 1.35 (3-H', m), 0.87 (5-H, d, 6.5), 0.85 (4-Me-H, d, 6.5). 13C NMR (125 MHz) 8 unit A 165.3 (1), 162.9 (12, d, 1Jc-F 245.4 Hz), 141.0 (3), 132.5 (9), 127.3 (10/14, d, 3JC-F
8.3 Hz), 125.3 (2), 115.7 (11/13, d, 2Jc-F 21.9 Hz), 76.1(5), 63.0 (7),.58.3 (8), 40.5 (6), 36.7 (4), 13.4 (6-Me); unit B 170.9 (1), 154.0 (7), 131.0 (5), 129.7 (4), 128.4 (9), 122.5 (6), 112.3 (8), 56.1 (OMe), 53.6 (2), 35.0 (3); unit C 175.6 (1), 41.1 (3), 38.2 (2), 14.1 (2-Me); unit D 170.7 (1), 71.3 (2), 39.4 (3), 24.5 (4), 22.9 (5), 21.3 (4-Me).
Cryptophycins 117 and 118 Using the general procedure described above for epoxidation of styrene-type cryptophycins, Cryptophycin 111 (6.2mg) was converted to 3.5mg of Cryptophycin and 1mg of Cryptophycin 118.
Spectral Data for Cryptophvcin 117 [a]D +25.5 (MeOH, c 1.8); EIMS m/z (relative intensity %) 668 (4.8, M+), 412 (6.2), 280 (11), 173 (9.4), 145 (15), 135 (34), 105 (100); high-resolution EIMS m/z 668.28532 (calcd for C36H45C1N208, A+1.1mmu, M+). 'H NMR (500 MHz) S unit A
7.17/7.13 (10-H/11-H/13-H/14-H, A2B2 q, 8.0), 6.67 (3-H, ddd, 15.4, 9.8, 5.6), 5.73 (2-H, dd, 15.4, 0.9), 5.14 (5-H, ddd, 11.2, 4.9, 2.0), 3.65 (8-H, d, 2.0), 2.91 (7-H, dd, 7.6, 2.0), 2.54 (4-H, bdd, 5.6, -14.3), 2.44 (4-H', ddd, 10.7, 9.8, -14.3), 2.35 (12-Me, s), 1.77 (6-H, m), 1.14 (6-CH3, d, 6.9); unit B 7.21 (5-H, d, 2.2), 7.07 (9-H, dd, 8.5, 2.2), 6.83 (8-H, d, 8.5), 5.65 (NH, d, 8.5), 4.80 (2-H, ddd, 8.3, 7.4, 5.6), 3.87 (OMe, s), 3.13 (3-H; dd, 5.6, -14.5), 3.02 (3-H', dd, 7.4, -14.5); unit C 6.93 (NH, bd, 6.8, 5.1), 3.48 (3-H, ddd, 5.1, 3.8, -13.2), 3.29 (3-H', ddd, 6.9, 6.8, -13.2), 2.71 (2-H, m), 1.22 (2-Me; d, 7.1); unit D 4.82 (2-H, dd, 9.8, 3.6), 1.70 (3-H/4-H; m), 1.33 (3-H', m), 0.85 (5-H, d, 6.5), 0.84 (4-Me-H, d, 6.5). 13C NMR (125 MHz) 8 unit A 165.3 (1), 141.0 (3), 138.4 (12), 133.7 (9), 129.4 (10/14), 125.6 (11/13), 125.3 (2), 76.2(5), 62.9 (7), 59.0 (8), 40.7 (6), 36.7 (4), 21.1 (12-Me), 13.6 (6-Me); unit B 170.9 (1), 154.0 (7), 131.0 (5), 129.8 (4), 128.4 (9), 122.4 (6), 112.3 (8), 56.1 (OMe), 53.6 (2), 35.1 (3);
unit C 175.5 (1), 41.1 (3), 38.3 (2), 14.1 (2-Me); unit D 170.7 (1), 71.3 (2), 39.4 (3), 24.5 (4), 22.8 (5), 21.2 (4-Me).

Spectral Data for Cryptophvcin 118 'H NMR spectrum is similar to that of Cryptophycin 38, except that the multiplets at 7.30-7.38 for the phenyl protons has been replaced by a 4H multiplet at 7.14 and a 3H
singlet at 2.35 for the p-tolyl protons.
CrYptophycins 119 and 120 Using the general procedure described above for epoxidation of styrene-type cryptophycins, Cryptophycin 112 (8mg) was converted to 1.2mg of Cryptophycin 119 and 0.5mg of Cryptophycin 120.
Suectral Data for Crvntophycin 119 'H NMR (500 MHz) S unit A 7.28 (12-H, d, 5.2), 7.11 (10-H, d, 3.3), 7.00 (11-H, dd, 5.2, 3.3), 6.68 (3-H, ddd, 15.1, 9.9, 5.2), 5.76 (2-H, dd, 15.1, 1.3), 5.15 (5-H, ddd, 11.2, 5.5, 1.8), 3.94 (8-H, d, 2.0), 3.10 (7-H, dd, 7.5, 2.0), 2.56 (4-H, bdd, 5.2, -14.3), 2.44 (4-H', ddd, 11.2, 9.9, -14.3), 1.76 (6-H, m), 1.13 (6-CH3, d, 6.8); unit B
7.21 (5-H, d, 2.3), 7.07 (9-H, dd, 8.4, 2.3), 6.83 (8-H, d, 8.4), 5.63 (NH, d, 8.4), 4.80 (2-H, ddd, 8.5, 7.4, 5.7), 3.87 (OMe, s), 3.13 (3-H; dd, 5.7, -14.5), 3.03 (3-H', dd, 7.4, -14.5); unit C 6.95 (NH, bd, 6.5, 5.3), 3.48 (3-H, ddd, 5.3, 3.6, -13.5), 3.31 (3-H', ddd, 6.8, 6.5, -13.5), 2.72 (2-H, m), 1.23 (2-Me; d, 7.3); unit D 4.85 (2-H, dd, 10.3, 3.5), 1.70 (3-H/4-H; m), 1.33 (3-H', m), 0.86 (5-H, d, 6.3), 0.85 (4-Me-H, d, 6.3).
CrMtophycins 125 and 126 Using the general procedure described above for epoxidation of styrene-type cryptophycins, Cryptophycin 124 (47mg) was converted to 26mg of Cryptophycin and 12mg of Cryptophycin 126.
Spectral Data for Cryptophvcin 125 [a]D +35.6 (CHC13, c=0.9); high-resolution EIMS m/z 688.2301 (calcd for C35H42C12N208, 0+1.7mmu). 'H NMR (500 MHz) 6 unit A 7.33 (11-H/13-H, dt, 8.5, 2.0), 7.18(10-H//14-H, dt, 8.5, 2.0), 6.67 (3-H, ddd, 15.1, 9.9, 5.2), 5.73 (2-H, dd, 15.1, 0.9), 5.15 (5-H, ddd, 11.0, 4.7, 1.5), 3.66 (8-H, d, 1.9), 2.87 (7-H, dd, 7.4, 1.9), 2.53 (4-H, m), 2.42 (4-H', ddd, 10.6, 10.5, -14.4), 1.78 (6-H, m), 1.12 (6-CH3, d, 7.0);
unit B 7.20 (5-H, d, 2.1), 7.06 (9-H, dd, 8.3, 2.1), 6.83 (8-H, d, 8.3), 5.72 (NH, d, 8.1), 4.79 (2-H, ddd, 8.3, 7.9, 5.4), 3.86 (OMe, s), 3.12 (3-H; dd, 5.4, -14.5), 3.02 (3-H', dd, 7.4, -14.5); unit C 6.97 (NH, bdd, 6.3, 5.5), 3.46 (3-H, ddd, 4.4, 4.1, -13.7), 3.22 (3-H', ddd, 7.0, 6.3, -13.7), 2.70 (2-H, m), 1.22 (2-Me; d, 7.2); unit D
4.82 (2-H, dd, 9.9, 3.4), 1.70 (3-H/4-H; m), 1.33 (3-H', m), 0.87 (5-H, d, 6.5), 0.84 (4-Me-H, d, 6.5). 13C NMR (125 MHz) 8 unit A 165.3 (1), 141.0 (3), 135.3 (12), 134.4 (9), 128.9 (11/13), 126.9 (10/14), 125.3 (2), 76.0 (5), 63.1 (7), 58.2 (8), 40.4 (6), 36.7 (4), 13.4 (6-Me); unit B 170.9 (1), 154.0 (7), 131.0 (5), 129.8 (4), 128.3 (9), 122.4 (6), 112.3 (8), 56.1 (OMe), 53.7 (2), 35.0 (3); unit C 175.6 (1), 41.0 (3), 38.2 (2), 14.1 (2-Me); unit D
170.7 (1), 71.2 (2), 39.4 (3), 24.5 (4), 22.9 (5), 21.3 (4-Me).
Spectral Data for Cryptophycin 126 1H NMR (300 MHz) unit A 6 7.26 (11-H/13-H, d, 8.1), 7.17 (10-H//14-H, d, 8.1), 6.70 (3-H, ddd, 15.1, 9.4, 5.3), 5.81 (2-H, d, 15.1), 5.14 (5-H, bdd, 10.0, 4.5), 3.57 (8-H, bs), 2.85 (7-H, bd, 7.6), 2.66 (4-H, m), 2.59 (4-H', m), 1.77 (6-H, m), 1.04 (6-CH3, d, 7.0); unit B 7.23 (5-H, bs), 7.08 (9-H, bd, 8.4), 6.83 (8-H, d, 8.4), 5.82 (NH, d, 6.8), 4.81 (2-H, ddd, 7.2, 6.8, 5.4), 3.86 (OMe, s), 3.14 (3-H; dd, 5.4, -14.1), 3.03 (3-H', dd, 7.4, -14.1); unit C 7.03 (NH, bt, 5.7), 3.47 (3-H, ddd, 4.0, 3.7, -13.1), 3.34 (3-H', ddd, 6.8, 6.4, -13.1), 2.72 (2-H, m), 1.24 (2-Me; d, 7.0); unit D
4.90 (2-H, dd, 10.0, 2.5), 1.74 (3-H/4-H; m), 1.45 (3-H', m), 0.91 (5-H, d, 6.5), 0.86 (4-Me-H, d, 6.5). 13C NMR (125 MHz) 6 unit A 165.4 (1), 141.3 (3), 135.6 (12), 134.1 (9), 128.8 (11/13), 126.8 (10/14), 125.2 (2), 76.8 (5), 63.3 (7), 55.6 (8), 40.8 (6), 36.7 (4), 13.4 (6-Me); unit B 170.9 (1), 153.9 (7), 131.0 (5), 129.8 (4), 128.4 (9), 122.3 (6), 112.2 (8), 56.1 (OMe), 53.7 (2), 35.0 (3); unit C 175.7 (1), 41.0 (3), 38.2 (2), 14.1 (2-Me); unit D
170.8 (1), 71.4 (2), 39.3 (3), 24.6 (4), 23.1 (5), 21.3 (4-Me).
Examnle 20 Synthesis of Cryptophycins 121-123 and 127 Compound AJ
To a solution of BOC-L_-leucine monohydrate (1.245g, 5mmol) in 30mL anhydrous CH2C12was added solid EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride)(0.53g, 2.75mmo1) under N2 with stirring at 0 C. After stirring at 0 C for 1.5h the mixture was concentrated below 5 C and the residue was diluted with 35mL cold - EtOAc and washed successively with two portions (lOmL) each of ice-cold solutions of 5% aqu. KHSO4, 5% aqu. NaHCO3 and brine. The organic phase was separated, dried (MgSO4) at 5 C and evaporated below 5 C. The residual oil was diluted with ice-cold THF (5mL) and added to a solution of compound K (295mg, 0.5mmo1) in ice-cold anhydrous THF (5mL). A few crystals of DMAP were then added to the mixture which was stirred and allowed to reach 25 C overnight. 5% aqu. NaHCO3 solution (5mL) was added and the resulting two-phase mixture was stirred vigorously at 25 C for 2h. EtOAc (4OmL) was added, the aqueous phase was extracted with additional EtOAc (2x 20mL) and the combined organic layers were washed with water and brine, dried (NaSO4), filtered and evaporated. The residue was filtered through a plug of silica gel with 25 %
EtOAC in hexanes to yield compound AJ as a colourless oil (385mg, 96% yield):
[a)D
13.6 (c= 0.63, CHCC13); EIMS m/z (rel. intensity) 805/803/801(M+, 1%), 568/570/572/574 (9/10/6/2), 523/525/527 (5/6/1), 418/420/422/424 (15/15/6/2), 341/343/345/347 (58/70/35/9), 307/309/311 (29/22/10), 224/227/228/229 (10/100/31/14), 208/210/211/2'12 (20/83/45/54); HREIMS m/z 800.2218 (C35H48 35C14N208, A-5.3mmu);
'H NMR (CDC13) unit A d 7.21-7.35 (Ph-H5; m), 6.77 (3-H; ddd, 6.7/6.7/15.3), 6.4 (8-H; d, 15.8), 6.05 (7-H; dd, 8.7/15.8), 5.9 (2-H; d, 15.3), 5.0 (5-H; m), 2.6 (6-H; m), 2.55 (4-H2; m), 1.17(6-Me; d, 6.7); unit B 8 7.22 (5-H; s), 7.05 (9-H: d, 8.1), 6.84 (8-H; d, 8.1), 6.55 (NH; d, 7.8), 5.0 (2-H; ddd, 5.5/7.1/7.8), 4.68-4.8 (CH2CC13;
ABq, 11.9), 3.86 (OMe; s), 3.2 (3-H; dd, 5.5/14.0), 3.06 (3-H': dd, 7.1/14.0); unit D S 4.8 (NH; d), 4.2 (2-H; m), 1.65 (4-H; m), 1.55 (3-H; m), 1.4 (CMez); s), 1.37 (3-H'; m), 0.85 (4-Me; d, 6.5), 0.8 (5-H; d, 6.5); 13C NMR unit A 8 165.4(1), 139.3 (3), 137.0 (9), 131.5 (8), 130.5 (7), 128.5 (11/13), 127.3 (12), 126.2 (10/12), 125.8 (2), 76.2 (5), 40.8 (6), 33.4 4), 16.8 (6-Me); unit B 8 170.0 (1), 154.2 (7), 131.0 (5), 129.0 (9), 122.5 (6), 112.2 (8), 94.4 (CC13), 74.7 (CH2CC13), 56.1 (OMe), 53.3 (2), 36.5 (3), unit D
8 173.2 (1), 155.7 (BOC-CO), 80.0 (CMe3), 52.4 (2), 41.2 (3), 28.3 (CMe,), 24.8 (4), 22.8 (4-Me), 21.6 (5).
Comnound AK
Compound AJ (115mg, 0.134mmo1) was dissolved in TFA (3mL) and left at 25 C
for 1 h. The solvent was removed and the residue was evaporated repeatedly from CH2C12 then from toluene to leave an amorphous solid. This solid was dissolved in anhydrous THF (5mL) and sufficient diethylisopropylamine was added to adjust the pH of the solution to pH 8-9 when an aliquot of the solution was spotted onto moist pH paper.
The solution was cooled to 0 C with stirring and a solution of compound AL in THF
(3mL) was added. To prepare compound AL compound Z (55mg, 0.27mmol) was dissolved in THF (3mL) and diethylisopropylamine (0.046mL, 0.27mmol) was added.
To this mixture, after cooling to -15 C, pivaloylchloride (0.033mL, 0.27mmol) was added dropwise and the solution was stirred at -15 C for 10 min and at 0 C for 20 min.
The resulting suspension was then transferred into the solution of compound AJ
in THF.
The resulting mixture was stirred at 0 C and allowed to reach 25 C overnight.
After 12 hrs at 25 C 5% aqu. NaHCO3 solution was added and the resulting mixture was stirred vigorously at 25 C for 1.5h. EtOAC (40mL) was added and the phases were separated.
The aqueous phase was extracted with additional EtOAC (2x lOmL). The combined organic extracts were washed with 5% aqu. NaHCO3 solution (2OmL), 5% aqu.

solution (20mL) and brine, dried (NaSO4) and evaporated. The residual oil was chromatographed over silica gel eluting with 35% EtOAc in hexanes to yield compound AK as a colourless foam (98mg, 83 % yield): [a]D -8.3 (c 0.88, CHC13); 'H NMR
(CDC13) unit A 8 7.28-7.35 (Ph-H4; m), 7.22(H-12;m), 6.75 (H-3; ddd, 6.4/6.4/15.4), 6.4(H-8; d, 15.9), 6.04 (H-7; dd, 8.6/15.9), 5.95 (H-2; d, 15.4), 5.0 (H-5; m), 2.6 (H-4; m), 1.1 (6-Me; d, 6.8); unit B 8 7.22 (H-5, d, 1.5), 7.15 (NH; d, 7.6), 7.05 (H-9; dd, 1.5/8.2), 6.85 (H-8; d, 8.2), 5.0 (H-2; m), 4.8-4.68 (CH CCl3; ABq, 12), 3.86 (OMe; s), 3.2 (H-3; m), 3.1 (H-3'; dd, 7.2/14.1); unit C
8 5.0 (NH; m), 3.2 (H2-3; m), 2.55 (H-2; m), 1.1 (2-Me; d, 7.1); unit D S 6.12 (NH;
m), 4.4 (H-2; m), 1.65 (H-4; m), 1.55 (H-3; m), 1.4 (H-3'; m), 0.86 (4-Me; d, 6.8), 0.81 (5-H; d, 6.8); 13C NMR (CDC13) unit A 8 165.8(1), 138.9(3), 131.5(8), 130.4(7), 128.6(11/13), 127.4(12), 126.2(10/14), 125.8 (2), 76.3(5), 33.6(4), 16.6(6-Me); unit B 6 170.2(1), 154.1(7), 136.9(4),131.2(5), 128.6(9), 122.3(6), 112.2(8), 94.4(-CC13), 74.5(CHZCC13), 56.1(OMe), 53.4(2), 36.6(3); unit C S 175.4(1), 156.3(BOC-CO), 79.5(OCMe3), 43.6(3), 41.3(2), 15.1(2-Me); unit D 8 172.6(1), 51.3(2), 40.5(3), 24.5(4), 22.7(4-Me), 21.5(5).
Cryptophvcin 121 Compound AK (73mg, 0.082mmo1) was dissolved in AcOH (3.5mL), activated Zn dust (400mg) was added and the resulting suspension was sonicated for 45 min.
After stirring at 25 C for an additional 1.5h the mixture was diluted with CH2C12 (5mL) and filtered through CeliteR. The solids were washed with additional CH2C12 (lOmL) and the resulting filtrate was evaporated. The residue, without further purification, was dissolved in TFA (3mL), kept, at 25 C for 1 h and the solvent was removed in vacuo. The residue was evaporated repeatedly from CH2C12, then from toluene until a colourless solid was obtained. This solid was dissolved in dry DMF (3mL), FDPP (43mg, 0.108mmol) was added followed by a sufficient amount of diethylis6propylamine to adjust the pH of the solution to pH 8-9 when an aliquot was spotted onto moist pH paper. After stirring at 25 C for 16 hrs, the mixture was diluted with ether (4OmL) and washed with 5%
aqu.
KHSO4 solution (2 x lmL), 5% aqu. NaHCO3 (15mL) and brine. After drying (Na2SO4) and evaporation the residue was purified by reversed-phase HPLC on C-18 silica (EconosilR C-18, 22x 250mm) eluting with CH3CN/water 65:35 at 6mL/min. The fraction eluting at tR 48 min was collected and evaporated to leave an amorphous solid (26mg, 50% yield): [a]D +46.5 (c 0.81, CHC13); EIMS m/z 637/639 (M+, 1%), 449/451 (2/1), 420 (5), 411/413 (7/5), 227/228 (9/7), 195 (10), 184 (15), (40/86/29), 155/157 (100/26), 91 (85), 69 (86); HREIMS m/z 637.2864 (C35 H44 06, A + 5.5mmu); 'H NMR unit A 6 7.21- 7.35 (Ph-H5; m), 6.75 (H-3; ddd, 4.2/10.8/16), 6.4 (H-8; d, 16), 6.04 (H-7; dd, 8.8/16), 5.75 (H-2; d, 16), 5.1 ((H-5; m), 2.55 (H-4; m; H-6; m), 2.35 (H-4'; m); unit B 8 7.18 (H-5; d, 2), 7.05 (H-9;
dd, 2.0/8.3), 6.85 (H-8; d, 8.3), 5.7 (NH; d, 7.2), 4.7 (H-2; ddd, 4.9/7.2/7.7), 3.86 (OMe;
s), 3.1 (H-3; dd, 4.9/14.4), 3.0 (H-3'; dd,7.7/ 14.4); unit C 8 7.25 (NH; m), 3.5 (H-3;
m), 3.4 (H-3'; m), 2.55 (H-2; m), 1.2 (2-Me; d, 7.2); unit D 8 5.8 (NH; d, 7.2), 4.4 (H-2; m), 1.55 (H-4; m), 1.38 (H2-3; dd, 7.2/7.7), 0.76 (4-Me; d, 6.6), 0.74 (H-5; d, 6.6):
13 C NMR (CDC13) unit A 6 165.1 (1), 141.9 (3), 136.8 (9), 131.7 (8), 130.2 (7), 128.5 (11/13), 127.3 (14), 126.1 (10/12), 125.1 (2), 76.5 (5), 42.3 (6), 36.3 (4), 17.2 (6-Me);
unit B 6 171.0 (1), 154.1 (7), 130.8(5), 129.6 (4), 128.4 (9), (122.5(6), 112.4 (8), 56.2 (OMe), 54.4 (2), 35.4 (3); unit C S 175.8 (1), 41.0 (3), 38.6 (2), 14.8 (2-Me); unit D S
173.2 (1), 51.1 (2), 40.9 (3), 24.7 (4), 23.4 (4-Me), 21.5 (5) Cry_ptophycins 122 and 123 To a stirred solution (0 C) of Cryptophycin 121 (20mg, 0.032mmol) in anhydrous CH2C12 (1.3mL) was added 99% mCPBA (17mg, 0.lmmol) in one portion. Anhydrous toluene (0.7mL) was then added and the resulting mixture was stirred at 25 C
for 72 h.
The solvent was evaporated in vacuo and the residual solid was purified by reverse-phase HPLC on C-18 silica (Econosil R C-18, 22x 250mm) eluting with CH3CN/water 65:35 at 6mL min '. Cryptophycin 122 (9mg, 44 %) eluted at tR 37.5 min and Cryptophycin (5mg, 23 % ) eluted at tR 40 min.

Cryptophycin 122 [a] D+35.0 (c 1, CHC13); EIMS m/z 653/655 (1.6/0.7, M+), 411/413 (20/5), 280/282 (39/19), 252/254 (13/8), 223/225/227 (19/10/23), 211/213 (18/6), (51/13), 184/186 (49/11), 176/168/169(20/16/21), 155/156/157 (95/59/42), (60/40/24), 135/135 (30/11), 129/131(40/29), 91(100); HREIMS m/z 653.2906 (C35 35C1 N3 07, A-3.8mmu); 'H NMR (d6-acetone) unit A 8 6.65 (H-3; ddd, 38/11.0/15.0), 5.9 (H-2; dd, 1.9/15.0), 5.25 (H-5; ddd, 1.9/4.9/11.5), 3.82 (H-8; d, 2.0), 3.0 (H-7; dd, 2.0/7.7), 2.65 (H-q.; dddd, 2.0/2.0/3.8/14.5), 2.4 (H-4'; ddd, 11.0/11.5/
14.5), 1.85 (H-6; dqd, 4.9/7.4/7.7), 1.1 (6-Me; d, 6.9); unit B 8 7.45 (NH; d, 7.9), 7.22 (H-9; dd, 2.0/8.4), 7.0 (H-8; d, 8.4), 4.45 (H-2; ddd, 3.6/7.9/11.2), 3.84 (OMe; s), 3.2 (H-3; dd, 3.6/14.5), 2.75 (H-3'; dd, 11.2/14.5); unit C 8 7.8 (NH; d, 8.8), 3.65 (H-3;
ddd, 3.3/8.8/13.2), 3.1 (H-3'; ddd, 2.1/2.1/13.2), 2.55 (H-2; m); unit D S 7.35 (NH; d, 8.1), 4.25 (H-2; ddd, 4.8/8.1/10.8), 1.65 (H-4; m), 1.45 (H-3; ddd, 5.1/10.8/13.7), 1.35 (H-3'; 4.8/9.0/13.7), 0.8 (4-Me/ H-5; d, 6.5); 13C NMR unit A S 165.9(1), 140.8(3), 138.6(9), 129.4(11/13), 129.2(12), 126.7(2), 126.6(10/14), 76.0(5), 63.9(7), 59.3(8), 41.2(6), 37.7(4), 13.9(6-Me); unit B S 171.9(1), 154.6(7), 132.5(4), 131.4(5), 129.0(9), 122.4(6), 113.3(8), 56.9(2), 65.4(OMe), 36.4(3); unit C 8 177.2(1), 41.3(3), 38.9(2), 15.7(2-Me); unit D 8 174.2(1), 51.7(2), 40.6(3), 25.3(4), 23.1(4-Me), 21.5(5).
Crvptophvcin 123 [a]D +25.2 (c 0.58, CHC13); 'H NMR (CDC13) unit A 8 7.21- 7.35 ((Ph-H5; m), 6.75 (H-3; ddd, 4.1/10.9/14.1), 5.85 (H-2; dd, 1.4/15.2), 5.25 (H-5; m), 3.6 (H-8; d, 2.0), 2.9 (H-7; dd, 2.0/7.8), 2.7 (H-4; m), 2.55 (H-4'; m), 1.75 (H-6; m), 1.05 (6-Me;
d, 7.1), unit B d 7.18(H-5;d,2.0), 7.05 (H-9; dd, 2.0, 8.5), 6.85 (H-8; d, 8.5), 5.95 (NH; d, 7.7), 4.7 (H-2;ddd, 4.9/7.7/8.1), 3.86 (OMe;s), 3.15 (H-3; dd, 4.9/14.5), 3.05 (H-3'; dd, 8.1/14.5); unit C 8 7.25 (NH; m), 3.55 (H-3; ddd, 4.6/8.7/13.3), 3.15 (H-3';
ddd, 3.0/3.1/13.3), 2.55 (H-2; m), 1.2 (2-Me; d, 7.3); unit D 6 6.0 (NH; d, 8.2), 4.45 (H-2; m), 1.6 ( H-4; m), 1.55 (H2-3; m), 0.88 (4-Me; d, 7.1), 0.87 (5-H; d, 7.1);
Crvntophvcin 127 Cryptophycin 122 (5mg, 0.0075mmo1) was dissolved in CHC13 (2mL) and cooled to -40 C under N2. Trimethylsilylchloride (0.02mL, 0.157mmo1) was added dropwise and the resulting mixture was stirred at -40 C for lh. The solvent was evaporated and the residue was filtered through a plug of silica gel with 15 % EtOH in diethylether to yield Cryptophycin 127 (4mg, 77% yield): [a]D +28.6 (c 1.16, CHC13); EIMS m/z (0.5; M+-HCl); 411(2); 182/184 (22/26); 153/155 (68/40); 135(31);
107/108/109(55/22/31); 91/92(100/30); 79/81(45/35); HREIMS m/z 653. 2841 (C35H4435C1N3O7, 0 2.6mmu); ' H NMR (CHC13) unit A 8 7.3-7.4 (Ph-H5; m), 6.75 (H-3;
ddd, 4.2/10.9/15.0), 5.8 (H-2; dd, 1.5/15.0), 5.2 (H-5; ddd, 1.9/9.9/9.9), 4.65 (H-8; d, 9.6), 4.0 (H-7; brd, 9.6), 2.65 (H-4; m), 2.48 (H-6; m), 2.35 (H-4'; ddd, 10.9/11.2/14.5), 1.02 (6-Me; d, 7.0); unit B S 7.18 (H-5; d, 2.2), 7.05 (H-9;
dd, 2.2/8.6), 6.85 (H-8; d, 8.6), 6.05 (NH; d, 7.7), 4.65 (H-2; ddd, 4.7/7.7/8.6), 3.86 (OMe; s), 3.15 (H-3; dd, 4.7/14.6), 2.9 (H-3'; dd, 8.6/14.6); unit C 8 7.25 (NH; brdd, 4.1/5.7), 3.4 (H2-3; m), 2.55 (H-2; m), 1.15 (2-Me; d, 7.5); unit D S 6.2 (NH;
d, 8.2), 4.45(H-2; m), 1.65 (H-4; m), 1.55(H2-3; m), 0.93 (4-Me; d, 6.8), 0.92 (5-Me;
d, 6.8);
13C NMR (CDC13) unit A S 165.4(1), 142.4(3), 138.8(9), 128.8(11/13), 128.2(12), 128.0 (10/14), 124.0 (2), 75.6(5), 73.9(7), 62.1(8), 38.6(6), 36.4 (4), 8.5(6-Me);
unit B S
171.1(1), 154.0(7), 130.8(5), 129.8(4), 128.2(9), 122.4 (6), 112.4 (8), 56.1 (OMe), 54.5(2), 35.4(3); unit C b 175.8(1), 41.1(3), 38.8(2), 14.8(2-Me); unit D S
173.2(1), 51.1(2), 40.9(3), 25.0(4), 22.8(5), 21.8(4-Me).

Example 21 Synthesis of Cryptophvcins 128 and 130-134 CrYptophycins 128. 130 and 131 A crude 2:1 mixture of 5.7mg of Cryptophycins 117 and 118 was dissolved in CHC13 (0.5mL) and treated with trimethylsilyl chloride (511L) at -60 C for 3 h.
Reversed-phase HPLC separated the resulting mixture of chlorohydrins into Cryptophycin 128, Cryptophycin 130, and Cryptophycin 131. Further purification by normal-phase HPLC gave pure Cryptophycin 128 (2.1mg).
Spectral Data for Clyptophycin 128 [a]0 +51.4' (CHC13, c 0.4); IR v. 3408, 3281, 2958, 1747, 1731, 1668, 1538, 1505, 1258, 1179, 1067, 910, 733 cm-'; EIMS m/z (relative intensity %) 668 (0.3, M+-HC1), 500 (1.2), 445 (4.2), 407 (6), 318 (12), 274 (17), 240 (33), 199 (29), 155 (31), 141 (23), 135 (15), 109 (100); high-resolution EIMS 668.2851 (calcd for C36H45C1N208, A+1.3mmu, M+-HCl). 'H NMR (500 MHz) 8 unit A A 7.27 (10-H/14-H, d, 8.0), 7.18 (11-H/13-H, d, 8.0), 6.69 (3-H, ddd, 15.2, 9.6, 5.4), 5.79 (2-H, dd, 15.2, 0.8), 5.10 (5-H, ddd, 11.0, 8.4, 1.7), 4.63 (8-H, d, 9.6), 3.99 (7-H, bd, 9.6), 2.67 (4-H, bdd, 5.4, -14.2), 2.49 (6-H, dqd, 7.6, 7.6, 1.7), 2.36 (4-H', td, 10.7, -14.2), 2.35 (12-Me, s), 1.04 (6-CH3, d, 7.1); unit B 7.23 (5-H, d, 2.1), 7.09 (9-H, dd, 8.5, 2.1), 6.84 (8-H, d, 8.5), 5.83 (NH, d, 8.6), 4.80 (2-H, ddd, 8.2, 7.5, 5.7), 3.87 (OMe, s), 3.16 (3-H;
dd, 5.6, -14.4), 3.00 (3-H', dd, 7.5, -14.4); unit C 6.94 (NH, bt, 5.8), 3.53 (3-H, ddd, 5.4, 4.1, -13.5), 3.24 (3-H', ddd, 6.9, 6.6, -13.5), 2.74 (2-H, m), 1.22 (2-Me; d, 7.3);
unit D 4.92 (2-H, dd, 9.9, 3.2), 1.75 (3-H/4-H; m), 1.47(3-H', m), 0.94 (5-H, d, 6.4), 0.92 (4-Me-H, d, 6.4). 13C NMR (125 MHz) 6 unit A 165.6 (1), 141.6 (3), 139.2 (12), 135.3 (9), 129.7 (10/14), 127.9 (11/13), 125.2 (2), 76.4 (5), 74.0 (7), 62.0 (8), 38.4 (6), 36.3 (4), 21.1 (12-Me), 8.6 (6-Me); unit B 171.1 (1), 153.9 (7), 131.0 (5), 130.0 (4), 128.4 (9), 122.4 (6), 112.2 (8), 56.1 (OMe), 53.6 (2), 35.0 (3); unit C 175.3 (1), 41.3 (3), 38.3 (2), 14.0 (2-Me); unit D 170.6 (1), 71.3 (2), 39.7 (3), 24.7 (4), 23.1 (5), 21.5 (4-Me).
Spectral Data for Cryptophvcin 130 1H NMR (300 MHz) S unit A 7.27 (10-H/14-H, d, 7.8), 7.16 (11-H/13-H, d, 7.8), 6.69 (3-H, ddd, 15.1, 9.6, 5.2), 5.75 (2-H, d, 15.1), 5.40 (5-H, ddd, 10.6, 3.0, 1.8), 5.05 (8-H, d, 5.9), 3.74 (7-H, ddd, 10.5, 5.4, 5.2), 2.59 (4-H, m), 2.55 (6-H, m), 2.37 (4-H', m), 2.33 (12-Me, s), 1.06 (6-CH3, d, 6.9); unit B 7.21 (5-H, bs), 7.07 (9-H, bd, 8.4), 6.83 (8-H, d,,8.4), 5.77 (NH, d, 6.6), 4.80 (2-H, m), 3.87 (OMe, s), 3.12 (3-H;
dd, 5.6, -14.4), 3.02 (3-H', dd, 7.2, -14.4); unit C 7.00 (NH, bt, 6.5), 3.47 (3-H, ddd, 4.3, 4.0, -13.3), 3.24 (3-H', ddd, 6.7, 6.6, -13.3), 2.72 (2-H, m), 1.23 (2-Me; d, 7.3);
unit D 4.80 (2-H, m), 1.70 (3-H/4-H; m), 1.42(3-H', ddd, 7.9, 4.7, -13.0), 0.91 (5-H, d, 6.4), 0.86 (4-Me-H, d, 6.4). 13C NMR (125 MHz) unit A 165.5 (1), 142.1 (3), 138.8 (12), 135.2 (9), 129.5 (10/14), 127.5 (11/13), 124.8 (2), 77.8 (5), 74.2 (7), 67.2 (8), 39.4 (6), 34.6 (4), 21.1 (12-Me), 12.3 (6-Me); unit B 171.0 (1), 153.9 (7), 131.0 (5), 129.5 (4), 128.4 (9), 112.2 (8), 56.1 (OMe), 53.6 (2), 35.0 (3)' unit C 175.6 (1), 41.1 (3), 38.3 (2), 14.1 (2-Me); unit D 170.3 (1), 71.5 (2), 39.5 (3), 24.6 (4), 22.7 (5), 21.6 (4-Me).
Spectral Data for Cryptophycin 131 1H NMR (300 MHz) S unit A 7.18 (10-H/11-H/13-H/14-H, s), 6.64 (3-H, ddd, 15.2, 9.8, 5.4), 5.70 (2-H, d, 15.2), 5.06 (5-H, bt, 9.1), 4.86 (8-H, d, 9.7), 4.06 (7-H, bd, 9.7), 2.54 (4-H, bdd, 5.2, -14.2), 2.37 (12-Me, s), 2.13 (4-H', m), 1.85 (6-H, m), 0.97 (6-CH3, d, 6.6); unit B 7.21 (5-H, d, 2.1), 7.07 (9-H, dd, 8.4, 2.1), 6.83 (8-H, d, 8.4), 5.66 (NH, d, 10.0), 4.79 (2-H, bq, 7.7), 3.87 (OMe, s), 3.14 (3-H; dd, 5.6, -14.4), 3.02 (3-H', dd, 7.2, -14.4); unit C 6.93 (NH, bt, 6.2), 3.51 (3-H, ddd, 5.0, 4.2, -13.5), 3.27 (3-H', ddd, 6.7, 6.4, -13.5), 2.73 (2-H, m), 1.23 (2-Me; d, 7.3);
unit D 4.86 (2-H, dd, 9.7, 3.4), 1.75 (3-H/4-H; m), 1.46(3-H', m), 0.94 (5-H, d, 6.0), 0.93 (4-Me-H, d, 6.4).
Cryptophycin 132. 133 and 134 A mixture of Cryptophycin 124 and the Z isomer (134mg, 0.199mmo1) dissolved in dichioromethane (6mL) was allowed to react with m-chloroperbenzoic acid (103mg, 0.598mmo1) at room temperature for 36 h. Phosphate buffer (lOmL, pH 8) was added to remove the chlorobenzoic acid generated during the reaction. After 30 minutes the aqueous layer was replaced with dimethyl sulfide (50 L) and a fresh sample of phosphate buffer (lOmL). Stirring was continued for 30 minutes. The organic layer was separated, the solvent evaporated and the residue dried under vacuum for 12 h. The crude mixture of predominantly Cryptophycins 125 and 126 was dissolved in CHC13 (5mL) and treated with excess trimethylsilyl chloride (50 L) at -60 C for 3 h. The solvent was evaporated and the residue purified on a reversed phase HPLC (Econosil ODS silica, 250mm x 22mm, 35 % H2O/CH3CN, 6mL/min) to give Cryptophycin 134 (tR 52.5 min, 9mg, 6%), partially pure Cryptophycin 133 (tR 61 min, 32mg, 22%) and crude Cryptophycin 132 (tR
67.5 min, 72mg). Further purification by normal-phase HPLC (Econosil silica, 250mm x 10mm, 56 % EtOAc/hexane, 3mL/min) gave pure Cryptophycin 132 (65mg, 45 %).
Spectral Data for Cryptophvcin 132 [a]D +60.1 (CHC13, c=1.1); EIMS m/z (relative intensity %) 688 (1.6, M+-HCl), 412 (10), 261 (11), 195 (57), 184 (28), 165 (28), 155 (92), 135 (85);
high-resolution EIMS m/z 688.23563 (calcd for C35H42C12N208, A-3.8mmu, M+-HCl). iH
NMR (500 MHz) 8 unit A 7.33 (10-H/11-H/13-H/14-H, s), 6.67 (3-H, ddd, 15.2, 9.9, 5.1), 5.79 (2-H, dd, 15.2, 1.1), 5.10 (5-H, ddd, 11.1, 8.1, 1.5), 4.63 (8-H, d, 9.5), 3.98 (7-H, bd, 9.3), 2.62 (4-H, bdd, 5.1, -14.2), 2.47 (6-H, dqd, 7.4, 7.4, 1.5), 2.40 (4-H', td, 10.8, -14.2), 2.35 (12-Me, s), 1.02 (6-CH3, d, 7.0); unit B 7.21 (5-H, d, 2.1), 7.06 (9-H, dd, 8.4, 2.1), 6.82 (8-H, d, 8.4), 6.04 (NH, d, 8.5), 4.74 (2-H, td, 8.0, 5.4), 3.85 (OMe, s), 3.13 (3-H; dd, 5.3, -14.4), 2.95 (3-H', dd, 7.8, -14.4); unit C 7.00 (NH, bt, 5.9), 3.48 (3-H, ddd, 5.0, 3.9, -13.4), 3.23 (3-H', ddd, 6.6, 6.3, -13.4), 2.71 (2-H, m), 1.21 (2-Me; d, 7.2); unit D 4.91 (2-H, dd, 9.7, 3.4), 1.75 (3-H/4-H; m), 1.45(3-H', m), 0.92 (5-H, d, 6.6), 0.91 (4-Me-H, d, 6.6). 13C NMR (125 MHz) 6 unit A 165.7 (1), 141.6 (3), 137.3 (12), 134.8 (9), 129.4 (11/13), 129.0 (10/14), 125.2 (2), 76.4 (5), 74.0 (7), 61.4 (8), 38.4 (6), 36.2 (4), 8.7 (6-Me); unit B 171.1 (1), 153.9 (7), 131.0 (5), 130.1 (4), 128.4 (9), 122.3 (6), 112.2 (8), 56.1 (OMe), 53.8 (2), 34.9 (3);
unit C 175.3 (1), 41.0 (3), 38.1 (2), 14.1 (2-Me); unit D 170.6 (1), 71.3 (2), 39.7 (3), 24.7 (4), 23.1 5 (5), 21.5 (4-Me).

Spectral Data for Cryptophycin 134 'H NMR spectram is similar to that of Cryptophycin 27, except that two multiplets at 7.31 (10/14) and 7.36-7.40 (11/12/13) for the phenyl protons has been replaced by two doublets at 7.26 (10/14) and 7.36 (11/13) for the p-chlorophenyl protons.
Example 22 Structure-Activitv Relationships (SAR) and In Vivo Evaluation The cytotoxicities of Cryptophycins 1, 3 and 8 and the new cryptophycins against the human tumor cell lines KB and LoVo are shown in Table 6. Cryptophycin 51 and Cryptophycin 3 show comparable cytotoxicities (IC50's 3.5-5.8 nM).
Cryptophycin 52 (IC50's 43-7OpM), however, is slightly less cytotoxic than Cryptophycin 1(IC50's 9-29pM), and Cryptophycin 55 (33-47pM) is slightly less cytotoxic than Cryptophycin 8 (IC50's 9-19pM). The cytotoxicity IC50's for Cryptophycins 117, 122, 125, 127, 128 and 132 are comparable with those for Cryptophycins 1, 8, 52, and 55; however, the data does not presently admit to more meaningful comparisons.
Cryptophycin 52 is active in vivo, but requires roughly three times the total dosage compared with Cryptophycin 1. The in vivo activity of Cryptophycin 52 against five solid tumors of murine origin and three human solid tumors is summarized in Table 7.
Similarly Cryptophycin 55 was active in vivo, but also required three times the total dosage compared with Cryptophycin 8. The in vivo activity of Cryptophycin 55 against six solid tumors of murine origin and four human solid tumors is summarized in Table 8.
The in vivo data appears to correlate with the in vitro data.
Cryptophycins 117, 125, 128 and 132 are active in vivo against a pancreatic adenocarcinoma of murine origin (Panc 03). Cryptophycins 117 and 128 appear to be more potent, that is they require smaller total doses, than Cryptophycins 1 and 8, respectively, whereas Cryptophycins 125 and 132 are less potent, that is they require higher total doses, than Cryptophycins 1 and 8, respectively. The data is summarized in Table 9.

T/C values that are less than 42% are considered to be active by NCI
standards;
T/C values that are less than 10% are considered to have excellent activity and potential clinical activity by NCI standards. Gross log kill is defined as T-C/3.2 Td where T is the median time in days for the tumors of the treated group to reach 750mg, C is the median time in days for the tumors of the control group to reach 750mg, and Td is the tumor volume doubling time (T.H. Corbett et al., Cytotoxic Anticancer Drugs: Models and Concepts for Drug Discovery and Development, pp 35-87; Kluwer: Norwell, 1992).
Gross log kill values of > 2.8, 2.0-2.8, 1.3-1.9, 0.7-1.2, and <0.7 with duration of drug treaunent of 5-20 days are scored + + + +, + + +, + +, + and - (inactive), respectively. An activity rating of +++ to ++++, which is indicative of clinical activity, is needed to effect partial or complete regression of 100-300mg size masses of most transplanted solid tumors of mice. Cryptophycin 52 shows T/C values ranging from 0 to 14% for murine tumors and 4.1 to 16 for human tumors and gross log kill values ranging from 1.1 to 2 for murine tumors and 0 to 3.2 for human tumors.
Cryptophycin 1 shows T/C values ranging from 0 to 27 % and log kill values ranging from < 1 to 2.
Cryptophycin 55 shows T/C values ranging from mostly 0 to 4.4 % and log kill values ranging from 2.1 to >4.6 (cures) for all but one trial, the Colon 26 experiment which showed a gross log kill value of 1.2. Cryptophycin 8 shows T/C values of mostly 0%
and log kill values > 2.8 (several cures) as shown in Table 10. Cryptophycins 117 and 125 appear to have T/C and gross log kill values that are comparable with those of other epoxide-type cryptophycins, viz Cryptophycins 1 and 52, whereas Cryptophycins 128 and 132 have T/C and gross log kill values that are comparable with those of other chlorophycin-type cryptophycins, viz Cryptophycins 8 and 55.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those of ordinary skill in the art in light of the teaching of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the claims.

Table 1. Cytotoxicity data for cryptophycins and semi-synthetic analogs.
Corbett/Valeriote assay data for 5-fluorouracil, etoposide (VP-16) and tazol are included for comparison.

Type of Cytotozicity (Differential in Zone Units) Corbett Valeriote KB IC50 LoVo IC50 Compound g/disk Assay' g/disk Assayb ng/mL ng/mL
1 12.5 E/T( > 400)' N 0.005 0.003 2 25 E/T( > 400) N 0.007 0.0002 3 25 E/T( > 400) N 0.3 0.5 4 20 E/T( > 400) N 1.3 0.5 2.9 E/T( > 600) N 0.02 0.02 6 250 I a100 a100 7 z750 a480 8 30 E/T( > 500) 30 N 0.0002 0.01 9 15 Not Deter-mined a100 a100 12 Z100 z100 14 1.8 3 5-FU 2.5 M/T( > 400) 2.5 LL( > 400) VP-16 5 L(350),T(530)d 5 LL(260) tazol 0.2 M/H/T(z400) 'L= leukemia selective (e.g. ZLt2IO-7c3s and ZLUW - Zxa>-250ZU) M= murine solid tumor selective (e.g. ZC38 - ZLI210 a250 zu) H= human solid selective (e.g. ZH: - ZLi2io OZ0 ZU) E= equally cytotozic towards leukemia and solid tumor cell lines (inhibition zones 2250 zu) T= tllmor selective (e.g. ZL1210 -ZLMLsZC38 -ZLMLs and ZE{s - ZLvt 2250 Zll I= inactive (inhibition zones <250) 6N= non-selective towards tumor (leukemia) and normal cell (CFU-GM) lines LL= lymphocytic leukemia selective (ZLUiQ - ZCSJ-GM i250 Zu) ML= acute myelogenous leukemia (AML) selective (ZAõL - ZcRj.GM a250 zu).
Selective against drug-sensitive and drug-resistant cell 'lines (ZC38 - ZLML,Z

ZLmL andZEõ-ZLm}.
Selective against drug-sensitive cxll lines only.

Table 2. In Vitro Cytotoxicity Data of Cryptophycins Cryptophycin KBIC50 LoVoIC50 SKOV3IC50 ng/mL ng/mL ng/mL
1 0.0025 0.001 0.026 2 0.023 0.021 0.18 3 1.8 0.6 2.8 4 6 2.5 21 12 2 7.4 8 0.01 0.0022 0.15 16 0.08 0.02 0.64 17 4.7 5.9 11 18 15 4.5 23 19 9.8 5.9 41 21 0.01 0.0003 0.029 23 0.89 0.4 1.7 24 0.12 0.095 0.3 26 19 9.8 95 28 1.5 0.75 6.1 29 1 0.49 3.4 31 0.53 0.062 1.9 35 0.055 0.01 0.092 40 9.0 1.0 1.7 43 0.72 0.8 1.1 45 2.3 2.4 1.6 49 1.4 1.9 1.1 50 0.17 0.17 0.2 54 0.80 2.2 2.2 Table 3. In Vivo Activity of Cryptophycin I

mgfkg % Body #. of Inj. Total . . Wt.
Exp # SC :Tumor : IV Dose Lossat T%C Log Cure Nadir Kill s 1560 Colon 38 8 10.3 Gain 6% 1.5 0/5 1694 Panc 03 8 16.0 Gain 0% 2.0 0/5 1636 Colon 51 7 28.1 -11% 7% 1.3 0/5 1720 Mam 16/C 5 13.2 -1% 5% 1.4 0/5 1733 Mam 16/Taxol 5 16.5 0% 2% 1.8 0/4 1833 M17/0 (Adr. 5 5.4 -10% 23% <1 0/5 Sens.) 1749 Panc 02 5 11.0 -5% 20% 1.1 0/5 1596 Human Sm Cell 6 7.3 0% 27% < 1 0/5 L.

SCID
1806 MX-1 Human 8 12 -3% 3% 2.0 0/5 Breast 1823 H125 Human 8 14.4 -15% 9% 1.1 0/5 Adenosq-lung 1/5 dead 1841 LNCaP Human 6 6.5 -6% 26% <1 0/5 Prostate Table 4. In Vivo Activity of Cryptophycin Analogs ~o ..
#Of, Inj. mgllt'..,Body:., :Exp Agent ;:SC IV g Tll:: :Wt. 'T/C ' L+vg Cure # 7Ytmo I:ass . . I~ill s; , r rtt:;
Nadir 1813 Cryptophycin-2 P03 10 37 -2% 44% <1 0/5 1843 Cryptophycin-3 P03 4 28/5 -9% 54% <1 0/5 1769 Cryptophycin-5 C38 15 45 -2% >10 None 0/5 0%
1825 Cryptophycin-8 P03 11 106 -6% 4% 4.6 0/5 1885 Cryptophycin-8 Mam 7 21.3 -4.5% 6% 2.5 0/5 1887 Cryptophycin-8 C38 6 30 -2% 0% 2.8 1/5 B
1900 Cryptophycin-8 Colon 9 67.5 -1% 7% 1.8 0/5 1843 Cryptophycin-15 P03 5 18 -7% 83% None 0/5 1878 Cryptophycin-16 P03 9 82 -1% 89% None 0/5 1813 Cryptophycin-21 P03 9 27 -11% 61% None 0/5 (1/5 dead) = 1843 Cryptophycin-35 P03 7 23 -2% 11 k 1.3 0/5 Table 5. Cytotoxicities of antimitotic agents for SKOV3 and SKVLB1 cells Cells were treated with varying concentrations of the compounds indicated below for 48 hours. Cell numbers were then determined as indicated in the Methods section and the IC50 for each compound was calculated. Values represent the mean SEM for three experiments.

Cell Line ' Compound SKOV3 SKVLB1 Resistance Factor IC50 (nM) Vinblastine 0.35 0.25 4200 1700 12,000 Taxol 1 0.4 8000 2000 8,000 Cryptophycins 0.007 f 0.002 0.60 f 0.19 86 Tabie S. lN Vlb CyMxkfly Dat o[CYyptoplq-t3os Sp LOVO
icso nM nM
1 0.0092 0.0104 3 3.1-4.6 1 Si-S.8 8 0.019 0.0091 51 3.5 5.2 52 0.043 0.070 53 42 5.5 33 0.033 0.047 57 0.064 0.27 58 0 048 0.20 81 5.5 5.7 90 0.34 0.30 97 3.6 5.7 110 4.6 5.3 111 4.8 3.2 112 5.4 6.2 115 0ACi9 0.048 117 0.036 0.031 118 2.3 1.2 119 4.4 6.1 120 4.6 6d 122 0m1 69 1?S 0.05 0.006 127 0.176 0.075 128 0.023 2.S
132 0.082 0.037 SUMMARY
A =
-r .. ... . : , s Log,o Tumor Day Totei Ooie .. ~r of YNt Loss~. TIC Pertlel . '' Complete';, Turtwr Ce9. Free Tumor Exp # SC Tumor mglkg inJ. ot NadEr IMr 96= Regressions : Regresaiorn- KiN
Cikes Free Actlvtty Human Tumors 2069* H116 Colon 30 4 -6.3% 4.1 215 0l5 2.4 015 84 +++
(Upstagedl * LNCeP Prostate 48 8 -4.896 6.9 5/5 115 3.2 0l5 66 + + + + (Upstaged) * OlbiPanc 48 9 -2.396 16 - - None 0f5 68 - >

N>1~', 2087* H125 Lung In)ectlons in pru9reas ~

Mouse Tumors N
2063 Mem 171Adr 24 12 -4.796 10 - - 1.6 0I5 18 ++
2065 Colon 261A 64 10 -14.0% 14 - - 1.1 015 24 +
2068* Colon 38 55 8 =2296 0 1/5 1l5 1.6 0l5 95 + + (Upategedl 2080 e Mem 161C 30 3 -1.6% 5 - - 1.2 1 f5 59 + +
2082 Mam 16/ClAdr 36 4 0% 2 - - 2 015 22 +++
2088* Penc 02 Iniecttona In progreu ~ In Progress rA

OTable 7 1 .

IN.VlVO ACTIVITY OF CRYPTOPHYCIN-55 SUMMARY

.% Body . Lopto : Turrwr Day Total Dose A~ of Wt. Loss TIC Parttal Complete.
Tumor Celt Free : Tumor Exp X SC Tumor mg/kg inj. at Nadir fn 96 Regressions Regressions Kill, Cures Free Activity Human Tumors 2066 =' TSU Prostate 298.5 10 =8.1 % 0 515 515 4.0 216 d 94 + + + +
(Upstaged) 2069" H 116 Colon 315 * 7 -10.4% 0 4/5 3/5 6.3 215 d 84 ++++
& (Upstagedl 157 5 7 =7.6% 8 4/5 0/5 3.6 Ol5 d 84 ++++

2072" LNCaP Prostete 300 12 .3.1% 0 515 515 5.9 315 d 77 ++++ y I (Upsteged) 200 5 =7.7% 0 4/5 315 6.0 0/6 d 77 ++++
N
2075'* Gbb Panc 320 8 0.0% 4.4 - - >2.0 1/5 d 68 + + + bi 2087 H125 Lung Injections In progress rn Mouse Tumors 2064"' Mem 161C 144 10 =7.596 0 - - 3.9 015 d 92 ++++
2065 Colon 26 260 10 =296 0 - - 1.2 015 d 24 +
2068 Colon 38 237 8 .13% 0 515 415 4.7 015 d 95 + + + + (Upstaged) 2070" Peno 03 402 6 =6.0% 0 - - > 4.5 5!5 d 84 ++++
270 6 =5.0% 0 - - >4.5 4/5 d 84 + + + +
~ { 1 180 4 Ø8% 0 -- - >4.5 4/5 d 84 ++++

2071 Mam 17/Adr 180 6 =4.7% 0 - - 2.1 0/5 d 18 +++
2082 Mam 161ClAdt 180 6 =1.7% 0 - - 2.4 015 d 22 +++ w 2088 Panc 02 injections in progress o ~ Produced 1/5 drug deaths ** Exp. In Progress Table 8 Evaluation of Cryptophycin Analogs Against Early Stage Panc 03 in BDFI Male Mice Total % Body Median Tumor Tumor No. of Dose Drug Wt. Loss Size on day 14 T/C Free Cage Agent Inj. IV mg/kg Deaths or Gain (day) (Range) In % Day 14 C 1 No Rx -- -- -- +6.8% (12) 830 (393-1458) -- 0/5 m 2 Crypto #117 1 7.0 3/4 -20.% (12) 0 (one mouse) 0% 1/4 3 1 6 13.5 1/4 -8.2 (13) 75 (0-256) 9% 1/4 4 1 6 6.75 0/3 -2.4 (8) 247 (80-415) 30% 0/3 M 5 Crypto #125 7 42 0/4 0% (10) 0(0-63) 0% 3/4 6 1 7 21 0/4 -1.6% (10) 101 (0-126) 12% 1/4 tA 7 1 7 10.5 0/4 0% (8) 295 (264-467) 35% 0/4 m tt1 8 Crypto #128 3 57 1/4 -15.2% (10) 0 (all zero) 0% 3/4 9 1 4 32.5 0/4 -5.7 %(6) 0 (all zero) 0% 4/4 7t~ Ll 1 4 16.25 0/4 0% (12) 0 (all zero) 0% 4/4 C
~ Crypto #132 6 180 0/4 -5.7% (10) 0 (all zero) 0% 4/4 1 6 90 0/4 -3.3 %(8) 0(all zero) 0% 4/4 ~ 1 6 45 0/4 1.7% (10) 304 (108-505) 37% 0/4 Tumors implanted day 0; Drug treatments started day 3.
As of Day 14 all the mice appear to be in good condition and gaining weight.
There should be no more drug deaths.
Table 9 =165-/N V/Vp ACTiVtTY OF CRYPTOPHYCIN-8 {R~~
t~ii Mna 03 11 106 -a 4 4.8 t1V5 7917 rana 03 12 QO .1 0 2.8 OJS
188iA Mate 16/C 7 213 -4.6 a 2.5 O/S
18878 Colen 88 a 80 +2 0 3.0 0!5 1954 Coloq 38 11 132 .8 0 >4.5 5/3 + 4 11 s8 .6 0 >4.5 415 1lQS Co1on 2B 10 100 .6.4 0 23 OJS
2007 Ovsry-135 6 50 3.5 0 >2 215 Id 20) 8 60 O >2 113 (d 20) 18K Aiiaat 17/Adr 8 53.1 -0.8 0 3.2 O!8 ! 36.9 =1.8 .0 2.6 0!5 1!!0 Mun 161C/Adr 7 iS .6 O 2.5 015 =001 rana 02 9 126 =3.5 0 >2 'Ub (d25) 1921 9 67.5 -6 18 1.8 015 1974 IN P388 L.ru1c 7 81 -1116 5096 8.8 2.3 015 1924 Colon 51 9 67.5 .6 7 2.2 015 1848 11pC-1 t=lttnan 11 50 .6.9 0 >4.5 315 4 Bsasst 11 34.2 -4.5 0 4.5 1J5 1852 K125 tkiman 8 98 +3.8 0 1.8 015 i Adeno. Sq. Isnv 8 57.6 +9.8 0 1.6 Ol5 1883 TSU Muinan 7 84.5 -3.4 0 >4 515 Praststa 7 66.5 -3.3 0 3.6. O15 1878 tCa Hwnn 8 96 +3.1 7.3 1.6 016 rrestsb 8 67.2 -F 3.Q 14.7 0.9 015 l880 Adv. itsoa 8 108 -6.7 E!5 PR's 3.8 015 iitsman H1i6 8 76 .6.7 .415 Mt's 3.5 0/5 Cdon 8 53 -39 1!s PB's 2.9 0/5 1278 Adv. Stsea 6 Q1 3.2 515 CR's 8.10 4/5 LKCsP Hunun 6 67 =1.4 5l3 CH's 7.10 4/5 Proststs 6 40.2 3.2 515 CR's 8.00 215 Llailted numbers ot tuenor prowths on wblch to carry out log WA calculation.

.wa~..

Table 10 References 1. Eglof, G., Organic Chemistry: An Advanced Treatise, Gilmar et al. (ed.), pp. 31-46, John Wiley & Sons (1943).

2. Kemp, et al., Organic Chemistrv, Worth Publishers, Inc. (1980). 5 3.
Patterson, G. M. L. et al., J. Phycol. 27:530-536 (1991).

4. Corbett, T.H. et al., Cytotoxic Anticancer Drugs: Models and Concepts for Drug Discovery and Development, pp 35-87, Kluwer Academic Publishers: Norwell, 1992.
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6. Schwartz, R.E. et al., J. Ind. Microbiol. 5:113-124 (1990).
7. Hirsch, C.F. et al., U.S. Patent 4,946,835, issued August 7, 1990.
8. Sesin, D.F., U.S. Patent 4,845,085, issued July 4, 1989.
9. Sesin, D.F. and Liesch, J.M., U.S. Patent 4,868,208, issued September 19, 1989.
10. Sesin, D.F., U.S. Patent 4,845,086, issued July 4, 1989.
11. Skehan, P. et al., J. Natl. Cancer Inst. 82:1107-1112 (1990).
12. Bradley, G. et al., Cancer Res. 49:2790-2796 (1989).
13. Endicott, J.A. et al., Ann. Rev. Biochem. 58:137-171 (1989).
14. Beck, W.T., Biochem. Pharm. 36:2879-2887 (1987).
15. Moscow, J.A. et al., J. Natl. Cancer Inst. 80:14-20 (1988).
16. Trimurtulu, G. et al., J. Am. Chem. Soc. 116:4729-4737 (1994).
17. Smith, C.D. et al., Cancer Res. 54:3779-3784 (1994).

Claims (26)

Claims:

1. A cryptophycin represented by the structure:

wherein Ar is a phenyl group, a phenyl group substituted with a C1-C5 alkyl group, a phenyl group substituted with a C1-C5 alkoxyl group, a phenyl group substituted with a halogen, a naphthyl group, a furyl group, an indolyl group, a pyrrolyl group, a pyridyl group, or a thienyl group;
R1 is a halogen, SH, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, or phosphate;
R2 is OH or SH; or R1 and R2 taken together form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring or a monoalkylphosphate ring; or R1 and R2 taken together form a double bond between C18 and C19;
R3 is a C1-C5 alkyl group;
R4 and R5 are H; or R4 and R5 taken together form a double bond between C13 and C14;
R6 is a benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, haloalkoxybenzyl, or dihaloalkoxybenzyl group;
R7 and R8 are each independently a C1-C5 alkyl group;
R9 and R10 are each independently H or a C1-C5 alkyl group; and X and Y are each independently O, NH or alkylamino, wherein the following structures are excluded:

2. The cryptophycin of claim 1, wherein R8 is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl.

3. The cryptophycin of claim 1, wherein R7 is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl.

4. The cryptophycin of claim 1, wherein R8 is methyl, R3 is methyl; X and Y
are not both O.

5. The cryptophycin of claim 1, wherein R3 is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl.

6. The cryptophycin of claim 1, wherein R9 is methyl, ethyl, propyl, butyl, isobutyl, pentyl or isopentyl.

7. The cryptophycin of claim 1, wherein R10 is methyl, ethyl, propyl, butyl, isobutyl, pentyl or isopentyl.

8. The cryptophycin of claim 1, wherein at least one of the groups attached to C3, C6, C10, C16, C17 and C18 have R stereochemistry.

9. The cryptophycin of claim 1, wherein at least one of the groups attached to C3, C6, C10, C16, C17 and C18 have S stereochemistry.

10. The cryptophycin of claim 1, wherein Ar is phenyl, R1 and R2 taken together form a double bond between C18 and C19, R3, R7 and R8 are methyl, R4 and R5 taken together form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R10 is hydrogen, X is oxygen, and Y is oxygen or nitrogen.

11. The cryptophycin of claim 1, wherein R1 and R2 taken together form an R,R-epoxide ring.

12. The cryptophycin of claim 1, wherein R1 and R2 taken together form an S,S-epoxide ring.

13. The cryptophycin of claim 1, wherein R4 and R5 taken together form a double bond between C13 and C14.

14. The cryptophycin of claim 1, wherein R4 and R5 are hydrogen.

15. The cryptophycin of claim 1, wherein R1 is halo, and R2 is hydroxyl.

16. The cryptophycin of claim 1, wherein R1 and R2 taken together form an episulfide ring.

17. The cryptophycin of claim 1, wherein Ar is phenyl, R1 and R2 taken together form an R,R-aziridine ring, R3, R7 and R8 are methyl, R4 and R5 taken together form a double bond between C13 and C14, R6 is 3-chloro-4-methoxybenzyl, R9 is isobutyl, R10 is hydrogen, and X and Y are oxygen.

18. A pharmaceutical composition useful for inhibiting the proliferation of a hyperproliferative mammalian cell comprising an effective amount of a compound of any one of claims 1 to 17 together with a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 18 further comprising at least one additional anti-neoplastic agent.

20. Use of the cryptophycin of any one of claims 1 to 17 for the production of a medicament for the treatment of a pathological condition caused by hyperproliferating mammalian cells.

21. Use of the cryptophycin of any one of claims 1 to 17 for the treatment of a pathological condition caused by hyperproliferating mammalian cells.

22. The use of claim 20 or 21 wherein the mammalian cells are human cells.

23. The use of claim 20 or 21 wherein the pathological condition is characterized by the formation of neoplasms.

24. The use of claim 20 or 21 wherein the neoplasms are selected from the group consisting of mammary, small-cell lung, non-small-cell lung, colorectal, leukemia, melanoma, pancreatic adenocarcinoma, central nervous system, ovarian, prostate, sarcoma of soft tissue of bone, head and neck, gastric, stomach, myeloma, bladder, renal and neuroendocrine.

25. The use of claim 24 wherein the gastric neoplasms are pancreatic or esophageal.

26. The use of claim 24 wherein the neuroendocrine neoplasms are one of thyroid, non-Hodgkin's disease and Hodgkin's disease neoplasms.